survival motor neuron: Biological Overview | References
Gene name - survival motor neuron
Cytological map position - 73A9-73A9
Function - RNA-binding protein
Keywords - presynaptic and postsynaptic neuromuscular synapse, biogenesis of small nuclear ribonucleoproteins (snRNPs), splicing factor
Symbol - Smn
FlyBase ID: FBgn0036641
Genetic map position - 3L:16,573,513..16,574,340 [-]
Classification - Survival motor neuron protein and Tudor domain
Cellular location - cytoplasmic and nuclear
|Recent literature||Borg, R. M., Bordonne, R., Vassallo, N. and Cauchi, R. J. (2015). Genetic interactions between the members of the SMN-Gemins complex in Drosophila. PLoS One 10: e0130974. PubMed ID: 26098872
The SMN-Gemins complex is composed of Gemins 2-8, Unrip and the Survival motor neuron (SMN) protein. Despite multiple genetic studies, the Gemin proteins have not been identified as prominent modifiers of SMN-associated mutant phenotypes. This study used of the Drosophila model organism to investigate whether viability and motor phenotypes associated with a hypomorphic Gemin3 mutant are enhanced by changes in the levels of SMN, Gemin2 and Gemin5 brought about by various genetic manipulations. Modifier effect is shown by all three members of the minimalistic fly SMN-Gemins complex within the muscle compartment of the motor unit. Interestingly, muscle-specific overexpression of Gemin2 was by itself sufficient to depress normal motor function and its enhanced upregulation in all tissues leads to a decline in fly viability. The toxicity associated with increased Gemin2 levels is conserved in the yeast S. pombe in which it was found that the cytoplasmic retention of Sm proteins, likely reflecting a block in the snRNP assembly pathway, is a contributing factor. It is proposed that a disruption in the normal stoichiometry of the SMN-Gemins complex depresses its function with consequences that are detrimental to the motor system.
|Borg, R. M., Fenech Salerno, B., Vassallo, N., Bordonne, R. and Cauchi, R. J. (2016). Disruption of snRNP biogenesis factors Tgs1 and pICln induces phenotypes that mirror aspects of SMN-Gemins complex perturbation in Drosophila, providing new insights into spinal muscular atrophy. Neurobiol Dis 94: 245-258. PubMed ID: 27388936
The neuromuscular disorder, spinal muscular atrophy (SMA), results from insufficient levels of the survival motor neuron (SMN) protein. Together with Gemins 2-8 and Unrip, SMN forms the large macromolecular SMN-Gemins complex, which is known to be indispensable for chaperoning the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). It remains unclear whether disruption of this function is responsible for the selective neuromuscular degeneration in SMA. This present study shows that loss of wing morphogenesis defect (wmd), the Drosophila Unrip orthologue, has a negative impact on the motor system. However, due to lack of a functional relationship between wmd/Unrip and Gemin3, it is likely that Unrip joined the SMN-Gemins complex only recently in evolution. Second, disruption of either Tgs1 or pICln, two cardinal players in snRNP biogenesis, results in viability and motor phenotypes that closely resemble those previously uncovered on loss of the constituent members of the SMN-Gemins complex. Interestingly, overexpression of both factors leads to motor dysfunction in Drosophila, a situation analogous to that of Gemin2. Toxicity is conserved in the yeast S. pombe where pICln overexpression induces a surplus of Sm proteins in the cytoplasm, indicating that a block in snRNP biogenesis is partly responsible for this phenotype. Importantly, this study shows a strong functional relationship and a physical interaction between Gemin3 and either Tgs1 or pICln. It is proposed that snRNP biogenesis is the pathway connecting the SMN-Gemins complex to a functional neuromuscular system, and its disturbance most likely leads to the motor dysfunction that is typical in SMA.
|Garcia, E. L., Wen, Y., Praveen, K. and Matera, A. G. (2016). Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy. Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy. RNA [Epub ahead of print]. PubMed ID: 27268418
Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. This study used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.
|Aquilina, B. and Cauchi, R. J. (2018). Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte. BMC Res Notes 11(1): 378. PubMed ID: 29895323
Spinal muscular atrophy (SMA) results from insufficient levels of the survival motor neuron (SMN) protein. Drosophila is conducive to large-scale genetic-modifier screens which can reveal novel pathways underpinning the disease mechanism. The ability of a large collection of genomic deletions to enhance SMN-dependent lethality was tested. To test this design, it was asked whether this study can identify loci containing genes identified in previous genetic screens. The objective was to find a common link between genes flagged in independent screens, which would allow exposing of novel functions for SMN in vivo. Out of 128 chromosome deficiency lines, 12 (9.4%) were found to consistently depress adult viability when crossed to SMN loss-of-function heterozygotes. In their majority, the enhancing deletions harboured genes that were previously identified as genetic modifiers, hence, validating the design of the screen. Importantly, gene overlap allowed flagging of genes with a role in post-transcriptional regulation of mRNAs that are crucial for determining the axes of the oocyte and future embryo. SMN was found to also be required for the correct localisation of gurken and oskar mRNAs in oocytes. These findings extend the role of SMN in oogenesis by identifying a key requirement for mRNA trafficking.
In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. The evolution of this machinery was studied using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the Drosophila contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. These data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction (Kroiss, 2008).
Splicing of pre-mRNAs is catalyzed by the spliceosome, a macromolecular machine consisting of a large number of protein factors and the uridine-rich small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6, and U5. The biogenesis of these particles occurs in a stepwise manner. First, nuclear-transcribed, m7G-capped snRNAs U1, U2, U4, and U5 are exported into the cytoplasm, where a conserved sequence motif in these RNAs (Sm site) serves as a binding platform for the seven Sm proteins B/B', D1, D2, D3, E, F, and G. As a consequence, a ring-shaped Sm core domain is formed. This domain is crucial for subsequent steps in the biogenesis of UsnRNPs, such as formation of the hypermethylated m2,2,7G cap and import of the assembled particle into the nucleus. At a yet to be defined step, additional factors are recruited to form the mature UsnRNP particles that function in splicing (Will, 2001; Kroiss, 2008 and references therein).
Previous studies have shown that Sm proteins bind spontaneously, albeit in a hierarchical manner, onto UsnRNAs in vitro). However, in cellular extracts, this process depends on ATP and the activity of the multisubunit SMN complex (Fischer, 1997; Liu, 1997; Meister, 2001). Recently, a systematic interaction study on the human SMN complex has established its basic architecture (Otter, 2007). A modular composition was deduced where the three factors SMN, Gemin2, and Gemin8 form the backbone of the entire complex. Onto this core, the peripheral building blocks Gemin3/4 and Gemin6/7/UNRIP bind to form the functional unit. In support of this modular architecture, Gemin-containing subcomplexes have been identified composed of SMN/Gemin2, Gemin3-Gemin5, and Gemin6/7/UNRIP (Kroiss, 2008 and references therein)
The SMN complex not only functions in the assembly of the Sm core domain, but also influences additional steps in the biogenesis pathway of UsnRNPs. One such step is the nuclear import of the assembled UsnRNP, which is mediated by the SMN complex (or parts thereof) in conjunction with the import factor importin β. In addition, specific UsnRNP proteins and the cap hypermethylase Tgs1 have been found in association of the SMN complex (Mouaikel, 2003). This observation indicates that the SMN complex coordinates various events during UsnRNP biogenesis by assuming the role of a binding platform for the respective assisting factors (Kroiss, 2008).
The multisubunit composition of the human SMN complex has impeded the mechanistic dissection of the UsnRNP assembly process. Thus, although RNA interference studies indicated essential roles of several Gemins in the assembly reaction, their precise contributions remain unclear. To facilitate mechanistic studies and to gain insight into the evolution of the SMN complex, genomic databases were mined for organisms that lack individual Gemins and hence may contain a simpler assembly machinery. Indeed, this was the case for different organisms including dipterans. Drosophila was chosen for further investigation because of the wealth of genetic resources. An affinity chromatography strategy has permitted the purification of an assembly-active complex composed of SMN and Gemin2 only. Remarkably, this complex not only facilitated assembly of the Sm core domain but also discriminates between cognate and noncognate RNAs. Thus, a combined bioinformatic and biochemical approach revealed that the assembly reaction requires only two core proteins in vitro, even though SMN complexes from most metazoans are of considerable complexity. It is speculated that Gemins 3-8 have been recruited to the SMN complex in the course of evolution to integrate assembly with additional steps in the biogenesis of UsnRNPs (Kroiss, 2008).
To understand how the UsnRNP assembly machinery has evolved, homology searches were performed for all Gemin proteins constituting the human SMN complex in genomic databases of a variety of organisms. Because of its diverse functions and its transient cytoplasmic interaction with the SMN complex, the UNRIP protein has been excluded from this analysis (Kroiss, 2008).
SMN and Gemin2 orthologs (termed Yab8p and Yip1p) but no other Gemins can be found in the fungus Schizosaccharomyces pombe. Importantly, both orthologs interact physically and may hence form a functional unit. In Saccharomyces cerevisiae, however, only the distantly related Gemin2 ortholog Brr1p, but no SMN ortholog, could be identified. Brr1p has been proposed to be an ortholog of human Gemin2. However, because of its limited homology to Yip1p this finding has been questioned. Taking advantage of the dramatically increased genome databases and novel search algorithms (PSI BLAST), Brr1p can be defined as the single significant homolog of human Gemin2 and S. pombe Yip1p. Because reciprocal searches further support this homology, Brr1p is the ortholog of human Gemin2. Thus, S. cerevisiae retained only one Gemin and hence is unlikely to form a functional SMN complex. Interestingly, like S. pombe, the plants Arabidopsis thaliana and Oryza sativa contained orthologs of SMN and Gemin2 only. It is therefore concluded that SMN and Gemin2 represent the most primitive and ancestral version of the SMN complex. Surprisingly, the genome of Dictyostelium discoideum, a facultative multicellular organism, encoded orthologs of Gemin3 and Gemin5. Given that D. discoideum is basal to fungi and metazoans, the bioinformatic data suggest that both Gemins have been lost during evolution in the fungi branch but were retained in metazoans. Interestingly, the presence of a Gemin5 ortholog in Ostreococcus tauri but its absence in land plants indicates an independent gene loss in this phylum. Moreover, it was found that SMN and Gemins 2, 3, 5, 6, 7, and 8 are present in the cnidarian Nematostella vectensis, a basic metazoan. Gemin4 first appeared in the sea urchin Strongylocentrotus purpuratus, whereas it is absent in all ecdysozoans under study. This suggests that Gemin4 has joined the SMN complex only recently in evolution, most likely with the appearance of deuterostomians. Consequently, it was found to be part of the SMN complex in vertebrates such as Danio rerio but also cephalochordates like Branchiostoma floridae and Ciona intestinalis (urochordates). Thus, plants and some fungi possess a core complex composed of SMN and Gemin2 only, whereas an elaborate SMN complex has developed only in animal branches by addition of Gemin proteins (Kroiss, 2008).
The bioinformatic data indicated an evolutionary trend in the animal kingdom toward a multisubunit SMN complex (see Evolution of an RNP assembly system). Interestingly, however, no orthologs of most Gemins were found in the dipterans Drosophila melanogaster and Anopheles gambiae although they were present in closely related Apis mellifera and Nasonia vitripennis. Further analysis was restricted to D. melanogaster in this study. Besides the known SMN ortholog (Miguel-Aliaga, 2000; Rajendra, 2008), a Gemin2 ortholog was found encoded by CG10419 and putative orthologs of Gemin3 (Dhh1) and Gemin5 (Rigor mortis). Dhh1 protein shows high conservation in the N-terminal DEAD box helicase domain but possesses a diverged C terminus. Rigor mortis displays moderate homology to Gemin5 over the entire protein length. A phylogenetic analysis revealed that both evolve significantly faster than their orthologs in other organisms. This released evolutionary pressure might indicate the emergence of a novel function or the loss of a common one for these factors. These data suggest that D. melanogaster possesses a much simpler SMN complex as compared with vertebrates (Kroiss, 2008).
To investigate whether Dhh1 and Rigor mortis have retained their function in the context of the D. melanogaster SMN (dSMN) complex, use was made of a novel epitope tag. This tag consists of the first 30 aa of human SMN protein, which are specifically recognized by the monoclonal antibody 7B10 (Meister, 2000). Importantly, competition with synthetic peptide comprising this epitope allows native elution of tagged proteins from this antibody. Because D. melanogaster SMN protein lacks these 30 aa, a plasmid was constructed allowing the expression and subsequent purification of a protein fused to this epitope (termed TagIt epitope) after stable transfection of Schneider2 cells. In a TagIt-Dhh1 affinity purification, only small amounts of dSMN protein could be detected under physiological conditions but not at salt concentrations exceeding 250 mM. Thus, Dhh1 is only weakly associated with dSMN. Similarly, the role of Rigor mortis was investigated. No binding of Rigor mortis to dSMN has been observed, arguing against a stable association of this protein with the dSMN complex. These data suggest that Rigor mortis either functions in UsnRNP core formation in a manner different from vertebrate Gemin5 or has completely lost its function in the pathway of UsnRNP biogenesis (Kroiss, 2008).
To gain detailed insight into the composition of the D. melanogaster SMN complex, a TagIt-dSMN-expressing Schneider2 cell line was generated. Importantly, TagIt-dSMN was incorporated into a high-molecular-weight complex that also contained Gemin2. This implied that the tagged dSMN protein engages in interactions similar to those of its endogenous counterpart. The SMN complex was affinity-purified from extracts by means of 7B10 affinity chromatography. Affinity-purified proteins were separated by SDS-PAGE under reducing and nonreducing conditions and identified by protein mass spectrometry and Western blotting. Whereas the tagged SMN protein and its interactor dGemin2 could be readily identified, neither Dhh1 nor Rigor mortis was found under the purification conditions applied in this study (Kroiss, 2008).
It is known that the human SMN complex consists of the core machinery (i.e., SMN and Gemins) as well as the transiently interacting substrates that are transferred onto the UsnRNA during assembly. These are the Sm proteins and some UsnRNP-specific proteins. Strikingly, the entire set of Sm proteins, namely SmB, SmD1 (gene snRNP69D), SmD2 (CG1249), SmD3, SmE (CG18591), SmF (DebB), and SmG (CG9742), was prominently present in the elution. Furthermore, the UsnRNP-specific factors U1 70KU2A', the U2B''/U1A ortholog SNF, and the ortholog of the U5 specific protein (CG4849) U5 116kD were found reproducibly in the purified complex. However, the abundance of these specific proteins varied among preparations and was often substoichiometric (Kroiss, 2008).
During UsnRNP assembly, the SMN complex physically contacts the UsnRNAs (Fischer, 1997). In vertebrates, this interaction has been proposed to be mediated, at least in part, by Gemin5 and to occur in the cytoplasm. Interestingly, despite the absence of Rigor mortis in the TagIt-dSMN complex, snRNAs U1, U2, U4, and U5 were specifically coprecipitated with dSMN and dGemin2 antibodies from total Schneider2 cell extract. Identical results were obtained when the SMN complex was purified from the cytosol, where SMN is predominantly localized. Hence, in D. melanogaster, the SMN complex is sufficient to recruit a set of substrate proteins similar to those in vertebrates. In addition, the complex interacts specifically with U snRNAs in the cytoplasm, which reflects a situation previously observed in Xenopus laevis oocytes (Kroiss, 2008).
Previous studies have indicated that Gemins interact within the SMN complex in a modular manner. Interestingly, homology searches for components of the SMN complex in a variety of organisms have recapitulated this finding on an evolutionary scale. The most simple SMN-containing complex is composed of SMN and Gemin2 only and can be found in unicellular organisms such as the fission yeast S. pombe and in plants. The next level of complexity is characterized by the appearance of Gemin3 in D. discoideum, thus predating the emergence of the Fungi/Metazoa clade. The absence of Gemin5 from genomes of fungi and land plants and its presence in the green algae O. tauri and in D. discoideum indicate independent secondary gene loss in fungi and plants. This may be due to a role of Gemin5 outside of the SMN complex, which is not retained in these organisms (Kroiss, 2008).
Only later in evolution at the level when first metazoans developed, the building block composed of Gemins 6, 7, and 8 was added to the set of the Gemin family. From this time on, organisms had the potential to express an SMN complex similar in architecture to the human one. The only component that was not present at that point was Gemin4, which can be found only in the genomes of deuterostomians. Thus, the data suggest that the SMN complex evolved by a blockwise addition of Gemins to an ancient core complex of SMN and Gemin2 in a manner corresponding to their mutual biochemical association (Kroiss, 2008).
In striking contrast to this overall evolutionary trend, a remarkable simplification of this complex in the dipterans was found A. gambiae and D. melanogaster. In these animals, no orthologs of Gemin4 were found, as expected, but also of Gemins 6-8 were absent. However, these latter Gemins were clearly present in hymenopterans. Although orthologs of Gemin3 and Gemin5 were found in dipterans, they show a significantly higher evolutionary rate in dipterans than in other clades. These computational findings have been experimentally challenged by a biochemical approach that has allowed isolation of an assembly-active SMN complex from D. melanogaster. Indeed, the composition of the complex was remarkably simple and consisted of SMN and Gemin2 as the only stoichiometric components. Dhh1 (Gemin3) bound to this core complex only at low salt concentrations, and Rigor mortis (Gemin5) was not present at all. The D. melanogaster SMN complex therefore equals its counterpart in S. pombe and plants although the function of SMN and Gemin2 orthologs in these organisms has not been demonstrated. It is conceivable that Dhh1 and Rigor mortis have adopted novel functions in a different context because they rapidly diverge from their ancestors. Consistent with this notion, a function of Rigor mortis (Gates, 2004) in ecdysone signaling has been described (Kroiss, 2008).
Despite the obvious simplicity of the SMN complex in D. melanogaster, this study has provided evidence that this unit is functionally related to the SMN complex of mammals. First, a set of UsnRNP-related substrates, namely the common Sm proteins, UsnRNP specific factors (U1 70K, U2A', U2B''/U1A, and U5 115K), and UsnRNAs were found to be part of the complex. Most of these factors have previously been shown to bind to SMN complexes of vertebrates (Meister, 2002). Second, affinity-purified dSMN complex mediate the assembly of the Sm core domain in vitro. Similar to the situation in humans, a strong dependence was found of UsnRNP core assembly on temperature but not on ATP (Meister, 2002). However, at present the possiblility cannot be ruled out that assembly of UsnRNPs in D. melanogaster cytosolic extracts requires ATP hydrolysis as observed for the same reaction in vertebrates (Kroiss, 2008).
The obvious simplicity of the assembly system of D. melanogaster allowed the reconstitution of the dSMN complex from recombinant proteins and the investigation of its mode of action. Interestingly, strong cooperativity was observed in Sm protein binding onto the complex. Heterooligomers D1/D2 and B/D3 had only little affinity for the complex, but binding was greatly enhanced in the presence of recombinant Sm heterooligomer E/F/G. Further studies are required to determine the precise binding sites of all Sm proteins on the SMN complex and to test the influence of arginine methylation on Sm protein binding. It was an open question why UsnRNP assembly is strictly dependent on the SMN complex in vivo even though this reaction is spontaneous in vitro. Assembly studies with the D. melanogaster SMN complex show that precise assembly of the Sm core domain on UsnRNA was possible only when Sm proteins were prebound to the SMN complex, whereas misassembly of isolated Sm proteins occurred under the same conditions. In addition, human SMN and Gemin2 are likewise sufficient to specifically transfer Sm proteins onto UsnRNA. Hence, these data and similar studies performed in vertebrates argue for a dual role of the SMN complex as an RNP assembler and chaperone (Kroiss, 2008).
From an evolutionary point of view, these findings raise the question why dipterans can afford a minimized assembly system, whereas apparently other branches in the animal kingdom require a multicomponent SMN complex. The most plausible explanation for this paradox is that Gemins 3-8 are not primarily involved in the assembly reaction per se but rather in other steps during the UsnRNP biogenesis. Thus, it is known that the human SMN complex integrates several steps in biogenesis, such as cap hypermethylation and nuclear import. It is speculated that these steps will occur in dipterans independent of the SMN complex and may hence allow for the omission of individual Gemins. Further studies will be needed to test whether this is indeed the case (Kroiss, 2008).
In conclusion, these studies have shown that the integration of bioinformatics and biochemistry can be used to analyze cellular pathways functionally and evolutionarily. Similar strategies may prove to be powerful tools in the analysis of even more complex systems such as the spliceosome (Kroiss, 2008).
Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. This study use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. Ectopic overexpression of the dead helicase Gem3(DeltaN) mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3(DeltaN) overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3(DeltaN), it is predicted that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3(DeltaN)-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3(DeltaN). Through enhanced knockdown, a requirement of Gemin2, Gemin3 and Gemin5 was uncovered for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead to a speculation that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo (Borg, 2013).
Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, this study shows that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. Previous studies found that SMN-containing U bodies invariably associate with P bodies. U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, these results suggest that SMN is required for the functional integrity of the U body-P body pathway, which in turn is important for maintaining proper nuclear architecture (Lee, 2009).
The current findings demonstrate that mutation of the U body component SMN causes disruption of P bodies and exhibits very similar phenotypes to those of P body mutants during Drosophila oogenesis. SMN has been shown to be ubiquitously expressed in all cell types, which correlates with its essential role in fundamental cell processes such as snRNP biogenesis and RNA splicing. However, the expression level of SMN is not uniform among different cell types. This study observe various levels of SMN within Drosophila ovaries. Although SMN is detectable in somatic cells, germline cells in egg chambers show much higher expression of SMN. The differential expression of SMN in somatic cells and germline cells may reflect the different activities of RNA metabolism in these cells. Alternatively, the high levels of SMN in germline cells may act as a store for subsequent use during embryogenesis (Lee, 2009).
Consistent with previous studies, it was found that SMN is mostly distributed in the cytoplasm of nurse cells and oocytes. The concentration of SMN in the ooplasm is similar to that in the nurse cell cytoplasm. However, the distribution of SMN is not uniform subcellularly. SMN is undetectable in the nucleoplasm with one exception -- Cajal bodies are enriched with SMN. In the cytoplasm, concentrated spherical structures known as U bodies can be detected above the bright cytoplasmic background (Lee, 2009).
The enrichment of SMN in the U body and the Cajal body is likely related to snRNP biogenesis since both organelles contain high levels of snRNPs. However, the pattern of SMN is not identical to the pattern of snRNPs. In the nucleus, snRNPs are enriched at sites other than the Cajal body, namely on the chromosomes and in structures that are believed to be sites of splicing, known as speckle. In the cytoplasm, snRNPs are only detectable in U bodies, whereas SMN staining is bright in both the cytoplasm and in U bodies. Overall, the levels of snRNPs in the nucleus are much higher than those in the cytoplasm, while the distribution of SMN is the reverse; higher in the cytoplasm than in the nucleus. Why do snRNP and SMN distributions differ from one another? It is possible that snRNP distribution reflects both snRNP assembly and snRNP activity, whereas SMN reflects only snRNP assembly. Alternatively, SMN may have a function in the cytoplasm that is independent of snRNP assembly (Lee, 2009).
There are four lines of evidence that support the hypothesis that U bodies and P bodies interact with each other. This study, consistent with previous observations, demonstrates that U bodies invariably associate with P bodies, although the absolute number of bodies may vary from cell to cell. However, P bodies are not always found to be associated with U bodies. This may be due to the significantly larger number of P bodies than U bodies in a cell. It is not known whether U body-free P bodies are functionally different from U body-associated P bodies. In some cases, many U bodies are clustered together surrounding one or more P bodies. The association of the U body with the P body is not disrupted even when the size and number of U bodies and/or P bodies change, as observed in many mutants. This suggests that there is a mechanism to maintain the connection between these two specific cytoplasmic domains that is not disrupted in mutants of P body components (Lee, 2009).
Secondly, smnA germline clones display similar nuclear morphology phenotypes to mutants in P body components. Analysis of combined mutations in U bodies and P bodies are underway to address the possible genetic interaction among the components in these two related cytoplasmic structures (Lee, 2009).
Next, it was previously shown that disruption of P body components such as Trailer-hitch or Ago2 leads to changes in U body organization. This study demonstrated that disruption of either one of two other P body components, Cup and Otu, leads to abnormal U body distribution and size. Collectively, these results indicate that P bodies are important for normal organization of U bodies (Lee, 2009).
Finally, using multiple P body markers, this study has shown that P body patterns are altered in cells in which the U body component SMN is disrupted. This suggests that factors in the U body can influence the structure of P bodies (Lee, 2009).
How does SMN regulate nuclear structure and function? It is hypothesized that SMN regulates nuclear organization through the U body-P body (Ub-Pb) pathway, where the U body and the P body, two specialized cytoplasmic domains, work together to regulate a series of downstream events including nuclear organization (Lee, 2009).
U bodies and P bodies associate and interact with each other, but remain physically and characteristically distinct from one another. The results from these experiments suggest it is likely that U bodies and P bodies are interdependent, and that components required by one may be regulated by machinery in the other, or vice versa. Moreover, the normal organization of the U body-P body association may simply reflect the balance between U bodies and P bodies. Any interference with this balance could impair the Ub-Pb pathway, which in turn, may lead to abnormal organization of U bodies and/or P bodies (Lee, 2009).
Both U bodies and P bodies are conserved structures in many cell types in multiple organisms. It would be particularly interesting to see how the Ub-Pb pathway works in other cell types such as neurons. Some human neuronal diseases are determined by factors in U bodies and P bodies. Factors that influence egg chamber development have also been shown to play key roles in the neuron, making the egg chamber an appropriate system in which to investigate the role of SMN. For example, low expression of SMN causes SMA, while Fragile X Syndrome is mainly determined by Fragile X Mental Retardation Protein (FMRP), a P body component. Indeed, a recent study has shown that SMN associates with FMRP in vitro and in the cell (Piazzon, 2008). It is hoped that these more detailed studies of U bodies and P bodies will give new insights into the subcellular and molecular mechanism of human diseases such as Fragile X Syndrome and SMA (Lee, 2009).
The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. This study describes the 2.5 Å crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis (Zhang, 2011).
Small nuclear ribonucleoprotein particles (snRNPs) are a major class of non-coding RNA-protein complexes that play key roles in post-transcriptional gene expression, including pre-mRNA splicing and suppression of premature termination. Each snRNP consists of one ~100-200 nucleotide small nuclear RNA (snRNA), called U1, U2, U4, U5, U11, U12, and U4atac, and a heptameric ring of Sm proteins (B/B', D1, D2, D3, E, F, and G) that surrounds the snRNA's Sm site (Sm core), as well as several proteins specific to each U snRNA. Sm cores are essential for the function, stability and nuclear localization of snRNPs, and their assembly is a key step in snRNP biogenesis. In vitro, purified Sm proteins can spontaneously form Sm cores on any RNA or oligoribonucleotide that contains a sequence resembling an Sm site, AUUUUUG or AUUUGUG. This assembly occurs stepwise from three Sm heteromeric subcore complexes, SmD1/D2, SmF/E/G and SmB/D3. SmD1/D2 and SmF/E/G first associate, forming a pentameric subcore that avidly binds RNA and this subsequently recruits SmB/D3, completing the formation of a highly stable Sm core. However, as sequences on which Sm proteins have the propensity to assemble do not uniquely define snRNAs, in cells potentially deleterious illicit Sm core assembly is prevented by the SMN complex, a molecular assembly machine that confers the necessary stringent specificity, ensuring Sm core assembly only on snRNAs (Zhang, 2011).
The SMN complex is comprised of SMN, Gemins 2-8 and Unrip. Recent findings showed that the active SMN complex, comprised of all of its known components, is made up of distinct subunits (Battle, 2007; Carissimi, 2005; Carissimi, 2006; Chari, 2008; Yong, 2010). The Sm proteins are recognized by a subunit that includes SMN and Gemin2 (Chari, 2008; Yong, 2010). The specificity for snRNAs is determined separately by Gemin5 (Battle, 2006; Lau, 2009), which recognizes a large ~50-60 nucleotide structure, called the snRNP code, that includes the Sm site and an adjacent 3'-terminal stem-loop structure found in all the pre-snRNAs and distinguishing them from other classes of RNAs. Additional subunits, one containing Gemins 6/7/8 and Unrip that can interact with SMN/Gemin2, and another comprised of the putative RNA helicase Gemin3 that can interact with Gemin4 and Gemin5, are also required for Sm core assembly in complex eukaryotes but their specific functions are not yet known. Despite significant advances, essential details on the process by which specific Sm core assembly is achieved in cells remain to be defined and a lack of atomic resolution structure of SMN complex components has limited progress on its mechanism and function. To date, only the structures of one domain in SMN, the Tudor domain, and of a Gemins 6/7 heterodimer have been described (Zhang, 2011).
This study identified Gemin2 as the protein that binds a pentamer of Sm proteins comprised of SmD1/D2 and SmF/E/G. The crystal structure of this complex bound to SMN's Gemin2 binding domain was determined to 2.5 Å, providing important mechanistic insights for SMN complex function and on Sm core assembly. An additional dimension of interest in the SMN complex and the snRNP assembly pathway comes from the fact that reduced levels of functional SMN, due to protein deficiency (>97% of the cases) or loss of function mutations, cause spinal muscular atrophy (SMA), a common motor neuron degenerative disease and a leading hereditary cause of infant mortality. Information from the structure that was determined explains the molecular basis of an SMA-causing patient mutation in SMN, linking a defect in Gemin2-mediated Sm pentamer recruitment to SMA (Zhang, 2011).
The structure and biochemical experiments provide mechanistic insights into the process by which the SMN complex assembles Sm cores and a structural basis for understanding the effect of an SMA-causing SMN mutation. Sm core assembly is a remarkable architectural feat requiring the seven Sm proteins to be brought together and form a ring around the pre-snRNAs' Sm site, a short nucleotide sequence present also in numerous other RNAs. To accomplish this, the SMN complex must gather the Sm proteins, inhibit their propensity for illicit Sm core assembly on unintended RNAs until a pre-snRNA joins. The current findings demonstrate that Gemin2 serves as the arm of the SMN complex that gathers five out of the seven Sm proteins, holding them as a pentamer poised for Sm core assembly and at the same time preventing them from binding RNAs. The structure explains how this is accomplished, revealing Gemin2 to be a key factor in snRNP biogenesis. Gemin2, through its extended conformation and remarkably extensive interactions with all five Sm proteins, grips the pentamer from its bottom and top sides, and from its outer parameter and inner pocket. Though its specific function was not previously known, Gemin2 has been shown to have a role in Sm core assembly. Consistent with this, the ubiquitously expressed Gemin2 is essential for viability of all eukaryotic organisms. Notably, Gemin2 gene deletion in the mouse causes embryonic lethality, at an even earlier stage than SMN gene deletio. Furthermore, Gemin2's sequence and domain structure are more phylogenetically conserved than that of all other SMN complex components, including SMN (Zhang, 2011).
The N-terminal tail of Gemin2, particularly residues 22-31, plays a role in inhibiting the pentamer from binding RNA as it occupies the pentamer's RNA-binding pocket. Furthermore, several residues in this part of Gemin2, including Met25 and Leu28 interact with the residues in the Sm proteins that are involved in binding Sm site nucleotides and are positioned in a way that would hinder RNA binding. Interestingly, these residues are conserved in Gemin2 orthologs from divergent organisms or are substituted by residues that are compatible with having the same activity, suggesting that this is a conserved function of Gemin2. The pentamer's narrower conformation in the Gemin2-bound state compared to that in the assembled Sm core would be expected to also restrict access of RNAs to the binding pocket. However, as the structure of an Sm pentamer alone is unknown, it is not possible to determine if Gemin2 binding plays a role in inducing or stabilizing the narrower conformation. Recent studies have shown that pICln, a protein that can bind Sm proteins and inhibit their interaction with snRNA, can bind at the SmD1-G opening, forming a closed hetero-hexameric ring that cannot bind snRNA. A complex suggested to represent a downstream intermediate, comprised of Drosophila C-terminal deleted SMN, Gemin2 and the Sm pentamer, which by electron microscopy shows a similar overall morphology to that of the current structure, has also been described (Chari, 2008). The data demonstrate that Gemin2 can bind the Sm pentamer and prevent it from binding snRNAs independent of pICln. Thus, there are at least two mechanisms of pentamer inhibition that are not incompatible and could occur sequentially, first by pICln, and subsequently by Gemin2. However, as pICln is not obligatory for Gemin2-pentamer association, it is also possible that the pentamer binds directly to Gemin2, which links the pentamer to SMN (Zhang, 2011).
For the subsequent steps of Sm core assembly to occur, after pre-snRNA is brought in by Gemin5, Gemin2's N-terminus, possibly up to α1, would need to be displaced from the Sm pentamer's RNA-binding pocket to allow the pre-snRNA to bind. The observation that Gemin2δN39 can bind the pentamer, suggests that such a displacement would not have the undesirable effect of dissociating the pentamer from the SMN complex. The SmD1-G opening and the Sm site-binding pocket would also need to be widened, utilizing the SmD2-F interface as a hinge. How these structural transitions are effected remains to be determined. Completion of Sm core assembly requires several additional steps and ATP hydrolysis, involving additional proteins about which little structural information is available. In complex eukaryotes, access of RNA to the inhibited intermediate, comprised minimally of SMN/Gemin2-Sm pentamer, is likely to be limited to only bona fide RNA substrates, pre-snRNAs, delivered by Gemin5. While it is clear that SMN is oligomeric in cells, the number of SMN subunits in a complex is unknown, bringing the possibility that it serves as a scaffold for more than one Gemin2-Sm pentamer forming on the same complex simultaneously. SMN determines the capacity of Sm core assembly (Wan, 2005) and its oligomerization is particularly important for this function as it serves to recruit essential components for this process. SmB/D3 association with the SMN complex is mediated at least in part by their direct interaction with SMN, which depends on SMN's oligomerization via its C-terminal YG-rich domains (residues 268-279) and in which the Tudor domain (residues 91-142) plays a role by binding to RG tails of SmB/D3 , an interaction that is strongly enhanced by arginine methylation that is carried out by the methylosome/PRMT5. There is evidence that an additional subunit that includes Gemins 6/7/8 and Unrip can also associate with SMN/Gemin2 and Sm proteins. Interestingly, Gemins 6 and 7 form a heterodimer and both have Sm folds and it has therefore been suggested that they might bind the pentamer in the same position where SmB/D3 bind, potentially forming a closed heptameric ring intermediate. This could further help maintain the pentamer's association with SMN/Gemin2, together with Gemin8 and Unrip. The function of Gemins 3 and 4, which exist as a dimer and associate with Gemin5, is not known, but the presence of a DEAD box domain in Gemin3 suggests that it may function as an RNA helicase and may be the source of the ATPase activity on which the assembly reaction depends. With the available structure of the key intermediate described in this study, several aspects of the mechanism and regulation of the SMN-Gemins complex as a molecular assembly machine for snRNP biogenesis can now be readily addressed (Zhang, 2011).
The structure further explains why D44V of SMN is an SMA-causing mutation. In the vast majority of SMA patients, the disease results from reduced levels of the SMN protein rather than from nonsense mutations. It is suggested that D44V is a loss of function mutation because it decreases the ability of SMN bearing this mutation to bind Gemin2 and thus impairs the SMN complex's capacity to recruit the Sm pentamer for snRNP assembly. These findings thus further link SMN's function in snRNP biogenesis to SMA. Further atomic level structural information could suggest approaches to enhance SMN-Gemin2 interaction as a potential therapy for SMA (Zhang, 2011).
Spinal muscular atrophy is a severe neurogenic disease that is caused by mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein is required for the assembly of small nuclear ribonucleoproteins and a dramatic reduction of the protein leads to cell death. It is currently unknown how the reduction of this ubiquitously essential protein can lead to tissue-specific abnormalities. In addition, it is still not known whether the disease is caused by developmental or degenerative defects. Using the Drosophila system, this study shows that SMN is enriched in postembryonic neuroblasts and forms a concentration gradient in the differentiating progeny. In addition to the developing Drosophila larval CNS, Drosophila larval and adult testes have a striking SMN gradient. When SMN is reduced in postembryonic neuroblasts using MARCM clonal analysis, cell proliferation and clone formation defects occur. These SMN mutant neuroblasts fail to correctly localise Miranda and have reduced levels of snRNAs. When SMN is removed, germline stem cells are lost more frequently. It was also shown that changes in SMN levels can disrupt the correct timing of cell differentiation. It is concluded that highly regulated SMN levels are essential to drive timely cell proliferation and cell differentiation (Grice, 2011).
This study shows a high demand for SMN in Drosophila stem cells. In addition, striking SMN concentration gradient, inversely proportional to the state of differentiation, has been identified in Drosophila larval CNS and testis. In Drosophila SMN mutant larvae, both the CNS and testis display growth defects which precede the previously reported motor defects and death. These larvae also fail to localise Miranda protein correctly at the basal membrane of the neuroblast. Clonal analysis indicates that SMN deficient stem cells have a reduced number of divisions and also generate cells with lower levels of U2 and U5 snRNPs. Overexpression of SMN alters the timing of CNS growth and disrupts the onset of pupariation and pupation. Using the male germline system, it was shown that prolonged SMN reduction leads to stem cell loss. Finally it was found that ectopic SMN expression in cells along the SMN gradient leads to changes in the timing of cell differentiation. It is therefore suggested that the fine-tuning of SMN levels throughout development can lead to complex developmental defects and reduce the capacity of stem cells to generate new cells in development (Grice, 2011).
SMN levels have been reported to be extremely high in early development. This study shows that SMN up-regulation occurs in neuroblasts prior to the initiation of their cell division, suggesting a distinct increase of SMN levels is required for new rounds of neurogenesis and local proliferation. Fewer immature neurons are generated in the thoracic ganglion of smn mutant MARCM clones. Provisional data has suggested there may be proliferation defects in the spinal cord of severe mouse models. In addition, a recent study using the severe SMA mouse model has shown proliferation defects in the mouse hippocampus, a region associated with higher SMN levels (Wishart, 2010). Together these data suggest that, in part, the pathology observed in more severe forms of SMA may be caused by defects in tissue growth (Grice, 2011).
Proteins involved in processes such as chromatin remodelling, histone generation and cell signalling have been identified as intrinsic factors for the maintenance of Drosophila stem cells. This is the first report of stem cell defects caused by the reduction of a protein involved in snRNP biogenesis. Although SMN is required in all cells, proper stem cell function requires a substantially higher level of SMN. This study also shows snRNP defects in Drosophila SMN mutant tissue. Previous studies in Drosophila have shown no gross changes in snRNP levels, including U2 and U5, in lysates from whole smnA and smnB mutant larvae. smnA MARCM neuroblast clones and male germline mitotic clones have reduced snRNP levels, suggesting snRNP assembly may be particularly sensitive to SMN reduction during CNS and germline development (Grice, 2011).
SMN mutant neuroblasts have abnormal Miranda localisation. Miranda, an adaptor protein, forms a complex with the RNA binding protein Staufen which binds to prospero mRNA. In addition to snRNPs, SMN protein has been implicated in the biogenesis of numerous RNP subclasses, including proteins involved in the transport and localisation of β-actin mRNA at the synapse. Whether Miranda mislocalisation is due to direct or indirect associations with SMN should be addressed (Grice, 2011).
SMN mutant larvae have been previously shown to have synaptic defects which include enlarged and fewer boutons and a reduction in the number of GluR-IIA clusters - the neurotransmitter receptor at the Drosophila neuromuscular junction. In addition, numerous developmental defects are observed including pupation and growth defects. Complementing this work, Drosophila Gemin5 a member of the Drosophila SMN-Gemin complex has been shown to interact with members of the ecdysone signalling pathway responsible for initiating pupation and growth. Drosophila Gemin5 is also enriched in pNBs, in a pattern comparable to SMN. There is increasing evidence that suggests the Drosophila SMN complex plays an important role in pupation. Ubiquitous overexpression of SMN using da-GAL4 advances CNS development and causes premature entry into pupation. The ecdysone pathway has been identified to play an important part in the regulation of neuroblast division and neuronal differentiation during development. How the Drosophila SMN complex plays a part in stem cell biology, and how the SMN complex interacts with specific signalling pathways should be the subject of further study (Grice, 2011).
Larval and adult testes exhibit the most distinct SMN gradients in Drosophila tissues. Drosophila testes have a constant population of germline stem cells that start to divide in the late larval stages and produce sperm throughout life. The removal of SMN from male germline stem cells results in stem cell loss. In the smnB mutant testis, the reduction of SMN causes a contraction of the SMN gradient towards the apical stem cells. As SMN is lost from the primary spermatoctyes, more mature sperm are observed. Increasing SMN levels leads to an increase in primary spermatocytes and a reduction in mature sperm in the adult. This result is the first to demonstrate that high SMN levels in undifferentiated cells can repress differentiation in sperm development. Interestingly, along with the CNS, Drosophila testes have the highest number of alternative splicing events and the most differentially expressed splicing factors during development. Understanding if differential expression of SMN in specific cell types controls a shift in splicing factors as cells switch from proliferation to differentiation will be the target of future study. A recent study has identified defects in gametogenesis and testis growth in mice lacking the Cajal body marker coilin, a binding partner of SMN. The authors speculated that coilin may facilitate the fidelity and timing of RNP assembly in the cell and coilin loss may limit rapid and dynamic RNA processing. It will be important to understand how SMN and coilin genetically interact in stem cells and developing tissues (Grice, 2011).
The Drosophila CNS and male germline offer two new tractable systems that can be used to study SMN biology in development and stem cells. It also offers a system to study how SMN, a protein associated with neuronal development, could cause SMA. Although SMA is classically a disease of the motor neuron, a severe reduction of SMN protein affects a wide spectrum of cells including stem cells. Consistent with this idea, symptoms in mild forms of SMA (type III or IV) are predominately limited to motor neurons. However, patients with the most severe type (type I), suffer from defects in multiple tissues including congenital heart defects, multiple contractures, bone fractures, respiratory insufficiency, or sensory neuronopathy. Elucidating the differential requirements of SMN in individual cell types, and how their sensitivity to SMN loss can mediate the disease, can contribute to the understanding of the selectivity of SMA (Grice, 2011).
Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, the fibroblast growth factor (FGF) signaling pathway was functionally linked to the Drosophila homologue of SMN, breathless with Smn. This study functionally characterize this relationship and demonstrates that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, it was shown that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities (Sen, 2011).
Given the variability of the SMA phenotype and the proven relationship between the severity of the disease and small changes in wild-type SMN activity, there is a significant possibility that any modifiers of SMN activity, either direct or indirect, will have therapeutic value. To systematically explore the genome for genes that are capable of modulating SMN function in vivo, advantage was taken of the existence of an SMA model offered by Drosophila to search for Smn genetic interactors. The model that was developed is based on the lethality and an associated neuromuscular junction phenotype linked to loss of Smn function, a phenotype remarkably similar to the NMJ phenotype reported for human patients. Though the role of SMN in biogenesis of snRNPs has been well documented, its regulators and downstream effectors have not been systematically delineated, nor has the link between mutations in SMN and the specific loss of motor neurons seen in SMA patients been uncovered. It may be the case that the specificity of this phenotype is reflective of either specialized SMN functions at the NMJ or a particular sensitivity of motor neurons to the loss of SMN activity. Among the genes the genetic strategy revealed as Smn loss of function modifiers was breathless, encoding an FGF receptor, thus establishing a link between Smn and the FGF pathway (Sen, 2011).
Importantly, in addition to this link, it was also found that FGF signaling is independently involved in NMJ morphogenesis, a function demonstrated in vertebrates but not previously attributed to this pathway in Drosophila despite extensive characterization of its essential role in branching morphogenesis of the tracheal system, migration of multiple cell types, as well as the proper patterning of the mesoderm. The morphological effects that were observed, caused by the modulation of several pathway elements, plainly reveal an involvement of FGF signaling at the NMJ, a role confirmed by the electrophysiological analyses. The down-regulation of FGF signals in muscle results in a reduction of bouton numbers and is associated with increased mEJP amplitudes. The opposite effect is observed when FGF signaling is increased in muscles, suggesting that FGF signaling inversely regulates quantal size. Thus, FGF perturbation in muscle alters both presynaptic growth and specific aspects of synaptic transmission. These observations imply the existence of functional trans-synaptic homeostatic mechanisms, which have been previously shown to compensate for similar changes by increasing presynaptic bouton numbers and transmitter release. However, in this specific instance, only synaptic growth (bouton number) but not transmitter release (quantal content) is affected, the precise mechanisms for which remain unclear. Moreover, the fact that mEJP amplitudes are affected suggests that postsynaptic receptivity to glutamate release from the presynapse is altered. Similar quantal size phenotypes have been observed in several instances previously. For instance, postsynaptic PKA and NF-kappaB are known to regulate quantal size through changes in DGluRs. Directly altering the expression of various GluR subunits also predictably influences quantal size. The genetic interaction this study has demonstrated between FGF and Smn can be described as an epistatic relationship in which the FGF pathway functions downstream of Smn and is consistent with the observation that neuromuscular defects associated with loss of Smn function in muscle can be rescued by muscle-specific activation of FGF signaling. Intriguingly, the relationship described in this study between Smn and FGF is valid beyond the NMJ, as loss of Smn function genetic mosaics in the wing disc clearly result in the down-regulation of FGF signaling. Although the precise molecular mechanism underlying this relationship is still elusive, Smn activity affects transcript and protein levels of the FGF receptor, as well as the expression of additional elements of the FGF pathway. Whether this defines a cascade of interrelated events or whether each of these changes reflects an independent Smn-related regulatory event remains to be determined. Given the fact that Smn mutants in Drosophila display altered postsynaptic currents and severely compromised postsynaptic receptor clustering in muscles, it is conceivable that FGF signaling represents a link between Smn activity and postsynaptic glutamate receptor levels (Sen, 2011).
It should be noted that a link between SMN and the FGF pathway has been suggested by a series of studies in vertebrates where a molecular interaction between an FGF-2 isoform and the SMN protein has been described.These studies raise the possibility that FGF-2 may negatively interfere with SMN complex function through SMN itself. Such observations would, on first appearance, suggest that the epistatic relationship between SMN and FGF signaling in vertebrate cells may be the reverse of what was observed in Drosophila. In point of fact however, the differences in the experimental parameters and approaches between these studies do not allow meaningful comparisons (Sen, 2011).
An important question raised by the above phenotypic analyses is whether the abnormalities associated with FGF and/or Smn perturbations reflect developmental or maintenance issues. It may be the case that the larval system in Drosophila is not ideally suited to differentiate between these alternatives as larval tissue is destined to undergo programmed cell death (histolysis) during metamorphosis. One advantage that flies do offer, however, is the ability to dissociate the development of the adult neuromuscular system from its maintenance as the entirety of its development occurs during the pupal stage, before emergence of the adult. Thus, the Drosophila pupa/adult may provide a platform to address these issues, as Drosophila displays Smn-dependent adult phenotypes. In light of the relationship that was established between Smn and FGF signaling and the known involvement of FGF signaling in the development of both the larval and adult musculature, it will be particularly interesting to examine the effects of modulating FGF activity on the aforementioned processes. Such studies may be of particular relevance to SMA where it is quite difficult to discern the developmental consequences of SMN loss in humans, as neurodegenerative symptoms displayed by patients may obscure basic problems resulting from altered developmental programs such as neuronal pathfinding, initial NMJ formation, etc (Sen, 2011).
In vertebrates, synaptic development and maintenance use at least three distinct signaling mechanisms: the TGF-β, wingless, and FGF pathways. In Drosophila, it is noteworthy that the first two have been demonstrated to function in a similar fashion at the NMJ. Remarkably, the genetic screens involving Smn have identified elements of all three of these pathways as modifiers of Smn-related phenotypes. These connections are considered particularly significant as they raise the possibility that Smn may serve as a node, integrating signaling events crucial for NMJ function, potentially leaving this structure particularly vulnerable to the loss of Smn. Though further correspondence between the Drosophila model and the human condition remains to be determined, the Smn-FGF relationship observed in Drosophila raises the possibility that pharmacological manipulation of FGF signals might mitigate SMN motor neuron-related abnormalities (Sen, 2011).
DEAD-box RNA helicase Gemin3 is an essential protein since its deficiency is lethal in both vertebrates and invertebrates. In addition to playing a role in transcriptional regulation and RNA silencing, as a core member of the SMN (survival of motor neurons) complex, Gemin3 is required for the biogenesis of spliceosomal snRNPs (small nuclear ribonucleoproteins), and axonal mRNA metabolism. Studies in the mouse and C. elegans revealed that loss of Gemin3 function has a negative impact on ovarian physiology and development. This work reports on the generation and characterisation of gemin3 mutant germline clones in Drosophila adult females. Gemin3 was found to be required for the completion of oogenesis and its loss led to egg polarity defects, oocyte mislocalisation, and abnormal chromosome morphology. Canonical Cajal bodies were absent in the majority of gemin3 mutant egg chambers and histone locus bodies displayed an atypical morphology. snRNP distribution was perturbed so that on gemin3 loss, snRNP cytoplasmic aggregates (U bodies) were only visible in wild type. These findings establish a conserved requirement for Gemin3 in Drosophila oogenesis. Furthermore, in view of the similarity to the phenotypes described previously in smn mutant germ cells, the present results confirm the close functional relationship between SMN and Gemin3 on a cellular level (Cauchi, 2012).
Severe reduction in Survival Motor Neuron 1 (SMN1) protein in humans causes Spinal Muscular Atrophy (SMA), a debilitating childhood disease that leads to progressive impairment of the neuro-muscular system. Although previous studies have attempted to identify the tissue(s) in which SMN1 loss most critically leads to disease, tissue-specific functions for this widely expressed protein still remain unclear. This study has leveraged RNA interference methods to manipulate SMN function selectively in Drosophila neurons or muscles followed by behavioral and electrophysiological analysis. High resolution measurement of motor performance shows profound alterations in locomotor patterns following pan-neuronal knockdown of SMN. Further, locomotor phenotypes can be elicited by SMN knockdown in motor neurons, supporting previous demonstrations of motor neuron-specific SMN function in mice. Electrophysiologically, SMN modulation in muscles reveals largely normal synaptic transmission, quantal release and trans-synaptic homeostatic compensation at the larval neuro-muscular junction. Neuronal SMN knockdown does not alter baseline synaptic transmission, the dynamics of synaptic depletion or acute homeostatic compensation. However, chronic glutamate receptor-dependent developmental homeostasis at the neuro-muscular junction is strongly attenuated following reduction of SMN in neurons. Together, these results support a distributed model of SMN function with distinct neuron-specific roles that are likely to be compromised following global loss of SMN in patients. While complementary to, and in broad agreement with, recent mouse studies that suggest a strong necessity for SMN in neurons, these results uncover a hitherto under-appreciated role for SMN in homeostatic regulatory mechanisms at motor synapses (Timmerman, 2012).
Although there are obvious differences between Drosophila and mammalian neuromuscular organization, modeling SMA in flies is a productive approach given the high degree of conservation in SMN function and the ease and rapidity of genetic analysis in flies. Indeed, previous work in Drosophila has suggested that Smn regulates U snRNP biogenesis and loss of SMN results in both synaptic and motor defects that are comparable to those seen in mouse models of SMA. Recent work has also made use of RNA interference to knockdown Smn in target tissues to better mimic the situation in SMA patients. Experiments with these validated RNAi reagents have uncovered morphological phenotypes at the NMJ when SMN was knocked down in either neurons or muscle. The current study used the same RNAi reagents in conjunction with the GAL4-UAS system to reduce SMN in neurons or muscle tissue followed by an assessment of such perturbation on motor performance and synaptic transmission at the NMJ. Muscle knockdown of SMN did not result in strong behavioral or synaptic transmission defects, though similar manipulations have been shown to affect synaptic morphology in larvae and lead to adult lethality. This suggests a situation in Drosophila that is very similar to that in mice, where SMN plays a role in muscles to ultimately impact lifespan, but is not critically required for normal synaptic functio. In contrast this study found distinct deficits in visuo-motor performance when SMN is reduced neuronally, though baseline synaptic transmission under these conditions is largely unaffected. This contrasts with earlier findings in Drosophila and is likely due to the compartment-specific knockdown of SMN in the current experiments. Interestingly, a recent report suggests that larval locomotor phenotypes in strong hypomorphic mutations in SMN arise from SMN deficiency in cholinergic neurons (Imlach, 2012). While the current complementary manipulations do not reveal any effect of cholinergic knockdown of SMN on locomotor activity in adult flies (perhaps due to insufficient knockdown with the available RNAi reagent), a very specific loss of homeostatic compensation between the pre- and post-synapse was identified when SMN was reduced neuronally. It is proposed that this might reflect an early and subtle event in SMA etiology that predates more dramatic SMA sequelae comprising loss of normal NMJs and neuro-muscular degeneration (Timmerman, 2012).
Severe mouse models of SMA (e.g., Smn-/-; SMN2+/+) show clearly discernible loss of neuro-muscular morphology, strong synaptic transmission defects, loss of pre-synaptic inputs to spinal motor neurons, muscle degeneration and eventual death of motor neurons. Several studies have also used Cre-loxP derived conditional knockout models of SMA in mice to address the question of tissue-specificity of SMN vis-a-vis the incidence of SMA-like symptoms. Complete removal of exon 7 in specific tissues led to cell lethality, a situation, though consistent with a vital role for SMN in all cells, significantly different from that found in SMA patients. Recent studies have more successfully approximated the patient condition, while trying to distinguish tissue-specific functions for SMN in vivo. In one study, Smn exon 7 was removed in neurons in a background where SMN2 was introduced. Surprisingly, these mice showed mild SMA symptoms that improved with age. However, this manipulation did uncover subtle homeostatic regulatory phenotypes at the neuro-muscular junction (NMJ) that may not have been noticed in the presence of the typically severe SMA phenotypes. In another study, full-length SMN protein was added back to either neurons or muscle in a mutant SMN background that otherwise results in viable 'SMA' mice. This study revealed that while both neuronal and muscle add-back of SMN could significantly rescue survival and motor behavior, only neuronal expression rescued synaptic function at the NMJ and also motor neuron somal synapses, through putative homeostatic mechanisms. A third study showed, by adding back SMN selectively to motor neurons using an Hb9-Cre, that SMN supplementation in motor neurons could rescue the vast majority of motor defects and restored normal sensory-motor synapses. Together, these observations hint at neuron-specific roles for SMN in the homeostatic regulation of normal synaptic connectivity and transmission in mammals (Timmerman, 2012).
Phenotypic end-points in severe SMA mouse models closely mirror SMA pathology in patients, particularly Type1 SMA. However, the progressive nature of these phenotypes, increased fetal expression of SMN and the general observation that SMA phenotypes can be most effectively rescued by adding back full length SMN perinatally, suggest that defining events in SMA pathology might occur at very early stages of development. Thus, obvious target tissues in SMA such as motor neurons and muscle might be exquisitely susceptible to a reduction in SMN function such that subtle defects in neuro-muscular development or physiology are the first to appear at these loci -- consistent with the threshold hypothesis for SMA. Milder models of SMA in mice seem to support this idea in some ways since they reveal aberrations in the homeostatic regulation of synaptic connectivity that may not be discernible in the presence of more severe phenotypes. These observations point to a fundamental impairment in communication between motor neurons and their pre-synaptic input following loss of SMN. Over time, this might lead to an asynchrony in an otherwise tightly coordinated program of synaptic development leading to eventual synaptic degeneration (Timmerman, 2012).
These experiments in Drosophila probably represent relatively mild manipulations of Smn. Consistent with this idea, animals are largely viable and do not show a dramatic loss of locomotion at larval stages that is seen in Smn loss-of-function mutants. However, neuronal knockdown of Smn does result in adult locomotor dysfunction that can be discerned with sophisticated visuo-motor assays. Further electrophysiological analysis at the larval NMJ provides a possible underlying mechanism, with the obvious caveat that these recordings are made at larval stages, while behavioral analysis is in adults. Although most parameters of synaptic transmission are near normal, specific phenotypes were detected when Smn is perturbed in neurons or muscles. Muscle knockdown of Smn results in altered quantal size. Although no changes were detected in glutamate receptor staining intensity or distribution, increased mEJP size might still result from altered post-synaptic receptor sensitivity as electrophysiological measures are typically more sensitive than immuno-histochemistry. Alternatively, altered quantal size might result from changes in synaptic vesicle size or neurotransmitter packaging, or from small changes in muscle size and input impedance. Neuronal knockdown of Smn leads to a defect in homeostatic compensation. A severe impairment of synaptic transmission as described previously, might not have allowed the detection of this phenotype. In general, this phenotype finds resonance with defects in homeostatic regulatory mechanisms that have been highlighted previously in mild SMA models in mice and might represent a 'weak link' that is most susceptible to a loss of SMN in neurons. It is also conceivable that an SMN-dependent loss of calibration in homeostatic drive is one of the earliest events that disrupts developmental coupling between the pre- and post-synapse during a critical period in synaptogenesis. It is interesting to note that this phenotype is only uncovered when Glutamate receptor expression is reduced, suggesting that low neuronal SMN limits the capacity for a homeostatic response when the demand for such a response is high. Further experiments are required to test whether this prediction holds in mouse models of SMA (Timmerman, 2012).
The current results might also help in understanding the molecular function of SMN in neurons. While SMN is clearly involved in U snRNP biogenesis, it is not clear whether this function of SMN is the one most compromised in neurons. For example, in Drosophila, Smn null mutants show normal mRNA splicing. In addition, low transgene-mediated expression of wild type SMN rescues SMA-like phenotypes in flies without any improvement in snRNA levels. By contrast, a recent report implicates aberrant U12 splicing events in a collection of U12 intron containing genes following SMN perturbations. While snRNA levels have not been tested in the current manipulations, phenotypic analysis shows clearly that a gene expression-requiring homeostatic compensation is completely abolished through a neuron-specific reduction in SMN. By contrast, a rapid gene expression-independent form of homeostasis at the NMJ is normal suggesting perhaps a role for SMN in the regulation of nuclear gene expression. Whether this occurs through control of mRNA metabolism, remains to be investigated. However, it is interesting to note that this form of long-term homeostasis also requires BMP/TGF-β signaling in the pre-synapse, a signaling pathway recently shown to interact with SMN in flies. In this model, one might envisage SMN playing a role either in the relay of a homeostatic 'signal' in the pre-synaptic compartment, or in the execution of a suitably graded response. Although these studies have previously shown a role for SMN in the regulation of FGF signaling in the muscle, this function does not seem to be necessary for homeostatic regulation of synaptic transmission. Finally, it is of note that motor neurons in Drosophila are glutamatergic. Whether GluR-dependent homeostatic signaling in flies has a bearing on cholinergic neuro-muscular synapses or glutamatergic sensory-motor synapses in mammals remains to be seen (Timmerman, 2012).
Spinal muscular atrophy (SMA) is a lethal human disease characterized by motor neuron dysfunction and muscle deterioration due to depletion of the ubiquitous survival motor neuron (SMN) protein. Drosophila SMN mutants have reduced muscle size and defective locomotion, motor rhythm, and motor neuron neurotransmission. Unexpectedly, restoration of SMN in either muscles or motor neurons did not alter these phenotypes. Instead, SMN must be expressed in proprioceptive neurons and interneurons in the motor circuit to nonautonomously correct defects in motor neurons and muscles. SMN depletion disrupts the motor system subsequent to circuit development and can be mimicked by the inhibition of motor network function. Furthermore, increasing motor circuit excitability by genetic or pharmacological inhibition of K(+) channels can correct SMN-dependent phenotypes. These results establish sensory-motor circuit dysfunction as the origin of motor system deficits in this SMA model and suggest that enhancement of motor neural network activity could ameliorate the disease (Imlach, 2012).
Across organisms, the function of the motor system seems uniquely sensitive to low levels of the ubiquitous protein SMN, the molecular defect responsible for SMA. This is also true in Drosophila, in which smn mutants have reduced muscle size and locomotion, which this study finds is accompanied by defects in rhythmic motor output and motor neuron neurotransmitter release. Surprisingly, restoration of SMN in the motor neurons or muscles of these mutants provided no phenotypic rescue. Instead, SMN must be reinstated in both cholinergic proprioceptive and central neurons to rescue smn mutant phenotypes, including non-cell-autonomous defects in both motor neurons and muscles. Proprioceptive neurons provide essential inputs to motor circuits, and cholinergic interneurons are critical for Drosophila CNS function, including synaptic output onto motor neurons. Restoration of SMN after the completion of nervous system development is sufficient to rescue SMN-dependent phenotypes, arguing that it is not the connectivity but rather the function of motor circuits that is disrupted by depletion of SMN. Two lines of evidence further support this conclusion. First, inhibiting the activity of cholinergic neurons can mimic a number of smn mutant phenotypes, including nonautonomous effects on motor neurons. Second, increasing the excitability of motor circuits through K+ channel inhibition can rescue smn mutant defects. The current results therefore demonstrate that depletion of SMN in Drosophila causes the dysfunction of a select subset of neurons in the motor circuit, which consequently disrupts the activity of other networked components of the motor system such as motor neurons and muscles. These findings establish this model of SMA as a paradigm for a neurological disease induced by neuronal circuit dysfunction (Imlach, 2012).
Although the results exclude a cell-autonomous requirement for normal SMN levels in Drosophila motor neurons to rescue smn mutants, the data do establish that SMN has to be restored in at least two groups of motor circuit neurons for full rescue of larval phenotypes. One of these groups is bd and type I md sensory neurons, which are essential components of a proprioceptive sensory feedback circuit necessary for coordinated contractile locomotion of Drosophila larvae. Both bd and type I md subsets of sensory neurons express the mechanosensitive NompC transcript receptor potential (TRP) channel, which is essential for proprioception. Sensory feedback does not seem to be necessary for Drosophila larval central pattern generator assembly or basic embryonic and larval movement; however, without sensory input, both rhythmic motor circuit activity and coordinated locomotion behavior are severely disrupted. Rescue of SMN in bd and type I md sensory neurons can restore the rhythmic motor output of smn mutants, which is consistent with an important role for sensory input in regulating this activity. However, restoration of SMN in proprioceptive neurons alone is not sufficient to correct the locomotion velocity of smn mutants, indicating that additional neurons require wild-type levels of SMN in order to restore full mobility (Imlach, 2012).
SMN expression in all cholinergic neurons can completely rescue all smn mutant larval phenotypes, including locomotion. The results therefore implicate an additional cell-autonomous requirement for SMN in one or more groups of central cholinergic neurons. Establishing the identity of these central neurons will be a challenge, given the limited understanding of central motor circuitry in Drosophila. It is tempting to speculate that these neurons could be descending inputs from the brain or other connections between segmental central pattern generators that promote the coordination necessary for effective locomotion. However, although rescue analysis demonstrates that individual components of the motor circuit can make significant contributions to some smn mutant phenotypes, other phenotypes such as muscle growth additively require SMN in both central and peripheral cholinergic neurons. Therefore, the data suggest that the effect of SMN depletion on the motor network is an amalgam of specific defects in distinct neurons that sum to produce a generalized disruption of the motor system (Imlach, 2012).
Why are cholinergic motor circuit neurons selectively susceptible to SMN depletion? In a companion manuscript (Lotti, 2012), a sequence of molecular events is described that link reduction of SMN to selective motor circuit dysfunction. Loss of Drosophila SMN disrupts minor splicing, which is required for the expression of genes with rare U12-type introns (see Janice, 2012). Through a genome-wide analysis of Drosophila U12 intron-containing genes, a transmembrane protein, Stasimon, was identified that has both reduced expression in smn mutants and increased NMJ eEPSP amplitudes when mutated, which is similar to the smn mutant phenotype. Like SMN, Stasimon is required in cholinergic neurons, but not in motor neurons, to affect NMJ electrophysiology. Furthermore, it was demonstrated that transgenic expression of Stasimon can fully restore normal NMJ eEPSP amplitudes in smn mutants in addition to increasing muscle size. These data establish that reduction of SMN decreases expression of a subset of genes that are particularly sensitive to SMN-dependent splicing disruption. Some of these genes, such as stasimon, are critically required for the normal function of cholinergic motor circuit neurons in Drosophila. These results establish a mechanistic chain linking the role of SMN in RNA splicing to the selective vulnerability of motor circuit function when SMN is depleted (Imlach, 2012).
Although the basic elements of motor circuits-proprioceptive neurons, interneurons, and motor neurons-are conserved between Drosophila and humans, the neuronal constituents and connections that make up Drosophila central motor circuitry are at present unknown, limiting comparisons with mammalian circuits. However, it is known that the neurotransmitters employed in each system are different. For example, human and mouse motor neurons are cholinergic, whereas proprioceptive neurons are glutamatergic, the inverse of the neurotransmitters employed in Drosophila motor circuits. Therefore, one possible interpretation of the current results is that cholinergic neurons have a particular and conserved sensitivity to the reduced levels of SMN. Neurotransmitter release is defective from the cholinergic motor neurons of SMN-Δ7 mice, and this defect does appear to require the cell-autonomous presence of normal SMN levels in these neurons. Nonetheless, SMN-Δ7 mutants have normal muscle twitch tension targeted depletion of SMN in motor neurons does not cause lethality, and selective restoration of SMN in motor neurons alone or cholinergic neurons alone produces only a few days of survival benefit to mutant animals. These results imply that, if indeed cholinergic neurons are selectively affected by reduction of SMN, additional neurons in the mammalian motor circuit must also be involved (Imlach, 2012).
An alternative, though not necessarily exclusive, interpretation is that conserved network elements of motor circuits are vulnerable to low levels of SMN. In support of this, it has recently been shown that SMN-Δ7 mice have early reduced responses to afferent fiber activatio, which are accompanied by a later decrease in glutamatergic proprioceptive synapses from sensory afferents onto motor neurons). SMA patients have also been reported to have reduced or absent H-reflexes, which could be consistent with decreased activity of motor reflex circuits. Interestingly, in a companion manuscript (Lotti, 2012), this study showed that the splicing and expression of the SMN-dependent gene Stasimon is preferentially disrupted in the proprioceptive neurons of SMN-Δ7 motor circuits, though motor neurons are also affected. The concordant evidence for defective sensory-motor function in both mammalian and Drosophila SMN mutants is striking but also unexpected, even with the limited understanding of the central circuitry of both systems. For example, both mouse and human motor neurons receive direct synaptic input onto both somata and dendrites from sensory afferents, whereas Drosophila motor neuron dendrites do not appear to contact proprioceptive axon processes (Zlatic, 2009). Restoration of SMN in the proprioceptive neurons of Drosophila smn mutants is sufficient to restore normal NMJ neurotransmitter release properties in motor neurons. This suggests that, even without direct synaptic contact, increasing SMN in these neurons can influence motor neuron electrophysiological properties, presumably through intermediate interneuron connections. Therefore, it is possible that, although the specific details of motor circuit wiring differ between Drosophila and vertebrates, the essential relationships and function of motor networks are conserved and selectively susceptible to depletion of SMN (Imlach, 2012).
Drosophila smn mutants have increased NMJ eEPSP amplitude and mEPSP frequency, which is consistent with an increased excitability of motor neurons. Hyperexcitability of motor neurons has also been described in the SMA-Δ7 mouse model. In Drosophila, this increase in neurotransmitter release properties is not corrected by restoring SMN in motor neurons themselves but is rescued by expressing SMN in cholinergic neurons. Hyperexcitability of Drosophila motor neurons has previously been reported in embryos in which cholinergic neurotransmission is completely inhibited. In agreement with this, the increased evoked neurotransmitter release from smn mutant motor neurons could be replicated by inhibiting cholinergic neurotransmission in larvae, which is consistent with a homeostatic compensatory increase in the excitability of motor neurons when synaptic inputs are reduced. A similar phenomenon has recently been described in chicken magnocellular neurons, which, when deafferentated by removal of the cochlea, increase in excitability. Increasing neuronal excitability by inhibiting K+ channels in smn mutants gave a remarkably robust rescue of muscle size, locomotion, rhythmic motor output, and NMJ neurotransmission. The Shaker type IA K+ current plays a critical role in the regulation of membrane excitability in Drosophila neurons, and expression of a dominant negative construct inhibiting the Sh current in cholinergic neurons of smn mutants fully rescues all the larval phenotypes examined. Together, these results strongly argue that decreased excitability of motor circuit neurons is a key physiological outcome of reduced levels of SMN (Imlach, 2012).
Treatment with the small-molecule K+ channel antagonist 4-AP also showed benefit to Drosophila smn mutant phenotypes. In wild-type animals, 4-AP treatment did not affect muscle size but did reduce locomotion and inhibited NMJ neurotransmitter release as might be anticipated by systemic inhibition of K+ channels, which are present throughout the nervous system and in muscles. Nonetheless, administration of 4-AP significantly increased both the muscle area and locomotion of smn mutants and fully corrected defects in rhythmic motor output and NMJ neurotransmission. Treatment with 4-AP has been linked to functional improvement of patients with spinal cord injury, myasthenia gravis, and Lambert-Eaton syndrome and can improve muscle twitch tension in a canine hereditary motor neuron disease. A sustained release preparation of 4-AP was recently approved by the FDA for human clinical use in multiple sclerosis. The data suggest that the efficacy of 4-AP in the Drosophila smn mutant model is likely via its activity upon cholinergic neurotransmission in the sensory-motor circuit. Extrapolating this finding to humans, investigation of compounds like 4-AP that can act within the spinal cord to modify the excitability of motor neural networks could be a fruitful therapeutic strategy to ameliorate the symptoms of SMA (Imlach, 2012).
Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, the role of SMN-dependent U12 splicing events (see Janice, 2012) was investigated in the regulation of motor circuit activity. SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA (Lotti, 2012).
Using mammalian cells and Drosophila larvae, this study demonstrates that the function of SMN in the assembly of spliceosomal snRNPs is required for efficient U12 splicing and that SMN deficiency decreases the expression of a subset of genes with this type of intron in vivo. While leaving open the possibility that SMN can also influence the activity of the U2-dependent spliceosome, as suggested by previous studies (Jodelka, 2010; Ruggiu, 2012; Zhang, 2008), the current findings establish direct regulation of U12 splicing events by SMN. Under normal conditions, U12 splicing is thought to represent a rate-limiting step in the expression of genes that contain U12 introns, which are processed more slowly than U2 introns (Patel, 2003). This, together with reduced availability of the snRNP components of the U12 spliceosome induced by low SMN levels, can explain the accumulation of SMN-dependent U12 splicing defects observed. Decreased mRNA levels of U12 intron-containing genes are likely due to degradation by surveillance mechanisms of incorrectly processed mRNAs resulting from SMN-dependent disruption of U12 splicing. In addition to increased U12 intron retention and decreased mRNA expression, exon-skipped and aberrantly spliced forms of some SMN target mRNAs also accumulate in SMN-deficient mammalian cells and, in the case of Stasimon, in the motor circuit neurons of SMA mice. Conceivably, these abnormalities are caused by poor exon definition consequent to inefficient binding of minor snRNPs to U12 introns (Lotti, 2012).
The results also highlight elements of selectivity in the effects of SMN depletion on U12 splicing. First, SMN deficiency causes selective rather than general defects in splicing, affecting some but not all of the U12 introns both in Drosophila larvae and mammalian cells. Second, the time of onset and degree of disruption in U12 intron splicing of SMN targets is variable and causes differential reduction of mRNA expression, which might have distinct functional consequences depending on the specific requirement of individual genes in vivo. Lastly, low SMN levels can affect evolutionarily conserved U12 introns of homologous genes in different species, pointing to a conservation of SMN splicing targets across evolution (Lotti, 2012).
To gain insight into the biological relevance of SMN-dependent U12 splicing events in motor circuit function, the role of SMN target genes on NMJ neurotransmission was investigated in Drosophila larvae. The results identify a U12 intron-containing gene, stasimon, which has reduced expression in Drosophila smn mutants and is required for the regulation of synaptic transmission of motor neurons. Decreased Stasimon activity elicits an increase in evoked neurotransmitter release at the NMJ. The effects of Stasimon deficiency on neurotransmission from glutamatergic motor neurons are caused by a dysfunction of cholinergic neurons in Drosophila. These findings indicate that the effects of loss of Stasimon on motor neurons are non-cell-autonomous and suggest that Stasimon is required for proper regulation of motor circuit activity (Lotti, 2012).
The results reveal a striking similarity in the effects of both Stasimon and SMN deficiency on the electrophysiological properties of Drosophila motor neurons. Evoked neurotransmitter release from motor neurons is increased in Drosophila smn mutants and can be corrected by transgenic SMN expression in cholinergic neurons (Imlach, 2012). The non-cell-autonomous increase in neurotransmitter release at the Drosophila NMJ is consistent with a hyperexcitable state of motor neurons resulting from reduced excitatory proprioceptive and interneuron input from the motor circuit. This is reminiscent of motor neurons in SMA mice in which an imbalance between excitatory and inhibitory inputs is correlated with a homeostatic increase in motor neuron excitability, presumably to compensate for the decreased presynaptic input. It is conceivable that similar events take place in the presence of reduced Stasimon function in the motor circuit (Lotti, 2012).
The precise mechanism by which reduced Stasimon perturbs motor circuit activity is presently unknown. However, Stasimon expression profile and protein structure suggest some possibilities. Stasimon is a ubiquitously expressed gene with a prominent expression in the Drosophila and mouse central nervous system and encodes a highly evolutionarily conserved protein containing six transmembrane domains and a region with homology to SNARE-associated Golgi proteins. These features are consistent with a neuronal function of Stasimon in transport or docking of vesicular cargo whose impairment in neurons could disrupt neuronal activity (Lotti, 2012).
SMN deficiency disrupts motor circuit activity in Drosophila. Importantly, proper regulation of motor circuit activity in Drosophila requires SMN function in cholinergic neurons, but not motor neurons (Imlach, 2012). The results reveal that restoration of Stasimon expression in cholinergic neurons is necessary and sufficient to fully rescue aberrant neurotransmitter release at the NMJs and to robustly improve muscle growth defects in SMN loss-of-function mutants, mirroring the cellular requirement for SMN in the Drosophila motor circuit. Therefore, decreased Stasimon function in cholinergic neurons directly contributes to disruption of motor circuit activity triggered by SMN deficiency and has non-cell-autonomous effects in motor neurons and muscle. These findings directly link selective neuronal effects of ubiquitous SMN deficiency to defective splicing of a gene with essential functions in motor circuits (Lotti, 2012).
The results further implicate Stasimon dysfunction in motor neuron phenotypes of a vertebrate model of SMA. SMN deficiency has been shown to elicit motor axon defects in zebrafish embryos. This study shows that Stasimon is required for normal motor axon outgrowth during zebrafish development and that Stasimon overexpression corrects the axonal defects in motor neurons with low SMN levels. Importantly, Stasimon does not rescue TDP-43-dependent motor neuron defects in a zebrafish model of ALS, providing evidence that Stasimon is a specific downstream target of SMN. Previous studies showed that the combined injection of major and minor snRNPs rescued motor axon defects in SMN-deficient zebrafish embryos, linking snRNP dysfunction to this phenotype. Based on these results, targeted ablation of minor snRNP components from the injected pool of snRNPs would be predicted to prevent correction of the motor neuron phenotype (Lotti, 2012).
The findings have important implications for understanding how mutations in ubiquitously expressed proteins cause the demise of selective neuronal types. Since the identification of SMN as the SMA-determining gene product and the discovery of its critical role in snRNP assembly, an unresolved problem has been how SMN-dependent snRNP biogenesis defects cause the selective dysfunction of the motor system. Particularly difficult to reconcile is the disruption of SMN ubiquitous activity in splicing with a selective pathology of motor function. The findings address this issue and provide evidence mechanistically linking disruption of SMN activity in snRNP assembly to motor neuron dysfunction through a cascade of molecular events with cause-effect relationships. First, SMN deficiency impairs Sm core formation, leading to a decrease in snRNP levels with effects that are tissue specific and particularly prominent on components of the U12 splicing machinery. Second, this reduction in snRNP levels causes selective splicing defects in a limited set of genes, resulting in alterations in their normal profile of expression. Third, a subset of these SMN target genes, including, but not necessarily limited to, those with U12 introns, performs functions that are critical for specific neuronal classes. Lastly, disruption of the activity of these genes, such as Stasimon, results in selective defects of neuronal function that collectively generate the SMA phenotype (Lotti, 2012).
Splicing defects have been thought to be too general to explain the highly discrete SMA phenotype, leading some to propose motor-neuron-specific functions of SMN. However, the current findings show that specificity can emerge through the combination of multiple mechanistic filters that act upon SMN’s ubiquitous role in splicing. The combination of these events accounts for the selectivity of the effects of SMN deficiency on the motor circuit in vivo. The results link SMN-dependent impairment of snRNP assembly to alterations in the expression of selected genes that cause motor neuron dysfunction, which is consistent with SMA being a disease of RNA splicing (Lotti, 2012).
Reduced levels of survival motor neuron (SMN) protein lead to a neuromuscular disease called spinal muscular atrophy (SMA). Animal models of SMA recapitulate many aspects of the human disease, including locomotion and viability defects, but have thus far failed to uncover the causative link between a lack of SMN protein and neuromuscular dysfunction. While SMN is known to assemble small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing, it remains unclear whether disruptions in splicing are etiologic for SMA. To investigate this issue, RNA deep-sequencing (RNA-seq) was carried out on age-matched Drosophila Smn-null and wild-type larvae. Comparison of genome-wide mRNA expression profiles with publicly available data sets revealed the timing of a developmental arrest in the Smn mutants. Furthermore, genome-wide differences in splicing between wild-type and Smn animals did not correlate with changes in mRNA levels. Specifically, it was found that mRNA levels of genes that contain minor introns vary more over developmental time than they do between wild-type and Smn mutants. An analysis of reads mapping to minor-class intron-exon junctions revealed only small changes in the splicing of minor introns in Smn larvae, within the normal fluctuations that occur throughout development. In contrast, Smn mutants displayed a prominent increase in levels of stress-responsive transcripts, indicating a systemic response to the developmental arrest induced by loss of SMN protein. These findings not only provide important mechanistic insight into the developmental arrest displayed by Smn mutants, but also argue against a minor-intron-dependent etiology for SMA (Garcia, 2013).
In summary, Smn-null larvae (harvested at 72-76 h post-egg laying) display significant locomotor deficits, but do not show appreciable genome-wide changes in mRNA levels. Based on morphological criteria, it has been previously concluded that Smn-null mutants are developmentally delayed. This study now shows by transcriptome profiling that Smn mutants display a molecular signature indicating a profound developmental arrest during the early third larval instar. Consistent with this notion, Smn-null larvae never display the wandering behavior that is characteristic of the late third instar, despite the fact that a small fraction of these animals live for several weeks. Proper staging of experimental and control animals is critical to any meaningful comparison (Garcia, 2013).
Transcriptome profiling reveals that Smn mutants display an increase in stress-responsive transcripts that may be a direct or indirect consequence of SMN loss. However, the role of these transcripts in the etiology of SMA-like phenotypes is not clear. The developmental arrest may be a downstream consequence of the inability of Smn-null larvae to feed properly. With this caveat in mind, the RNA-seq data provide evidence for the activation of stress signaling pathways that could represent an important systemic response to dSMN depletion. The activation of cellular stress pathways was previously observed in a mouse embryonic stem (ES) cell model of SMA and in spinal cord tissues from which these ES cells were derived. Thus, activation of stress- response pathways may be a conserved feature of SMA. Neuronal overexpression of certain antimicrobial peptides, which are known stress signaling targets, was recently shown to cause neurodegeneration in Drosophila. Taken together, these observations suggest that the activation of cellular stress signaling pathways in Smn mutants is an important pathophysiological consequence of SMN depletion that is relevant for modeling SMA (Garcia, 2013).
Overall, the observed developmental arrest complicates determination of cause and effect in the pathophysiology of Smn mutants. Both fruit fly and mouse models of severe SMA display neuromuscular deficits that are detectable early in the disease course, whereas splicing defects are only apparent later on. The RNA-seq data reveal several differences in pre-mRNA splicing between wild-type and Smn animals. However, these splicing differences do not correlate with changes in mRNA levels. The impact, if any, of these small changes on the organism's neurophysiology remains to be elucidated. Results presented in this study cannot exclude the important possibility that tissue- and cell-specific splicing events below the current level of detection might be causative for SMA. Future studies of mRNA splicing and abundance in isolated cells and tissues may uncover a more direct link between SMN depletion, splicing, and SMA-like phenotypes. Nevertheless, the results presented in this raise serious doubt as to the validity of conclusions reached by others regarding the connection between splicing deficiencies and motor circuit dysfunction. Clearly, additional studies will be needed in order to fully elucidate the molecular etiology of SMA (Garcia, 2013).
Mutations in the human survival motor neuron 1 (SMN; see Drosophila Smn) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, an allelic series was created of twelve Smn missense mutations, originally identified in human SMA patients. Animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN (Praveen, 2014 - Open access: 25144193).
Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. This study used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Normal neuromuscular junction (NMJ) structure depends on SMN expression, and SMN concentrates in the post-synaptic NMJ regions. A screen was conducted for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that the genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets (Chang, 2008. Full text of article).
Previous studies based primarily on the analysis of the Smn73Ao allele demonstrated that reduced Smn activity causes lethality and NMJ morphological defects (Chan, 2003). These observations were corroborated through the examination of several extant and novel Smn mutations of varied severities, including several GAL4-inducible Smn RNAi alleles generated for this study. These hypomorphic strains reduce SMN expression levels to different degrees in a manner formally analogous to decreased SMN levels observed in SMA patients. Additionally, these strains may model the dosage-dependent nature of SMA as the developmental arrest associated with these flies correlates with the extent of morphological abnormalities observed at the NMJ (Chang, 2008).
Examination of Smn NMJ structure in Drosophila using pre- and post-synaptic markers, SYT and DLG, respectively, revealed significant losses of synaptic bouton numbers in multiple Smn backgrounds. Moreover, in these backgrounds, reduced post-synaptic GluRIIA expression was detected, consistent with previous analyses of the Smn73Ao NMJ (Chan, 2003). Together, these results suggest that loss of SMN function in Drosophila causes aberrant neuromuscular synaptic structure, mimicking the pathology of SMA. In addition, these structural abnormalities are consistent with the altered electrophysiological profile previously observed in Drosophila Smn73Ao animals (Chan, 2003). It should be noted that other glutamate receptor subunits display altered transcriptional profiles in a Smn73Ao background; specifically the GluRIIA and GluRIIB transcript levels were decreased while GluRIIC levels were increased (Lee, 2008). Therefore, combining genetic and morphological analyses of pathological changes in synaptic structure with future electrophysiological studies will be necessary to understand more thoroughly the synaptic consequences of SMN loss in SMA (Chang, 2008).
A longstanding question in the pathology of SMA is the relative neuronal and muscle contribution of SMN function. The RNAi strains allowed reduction of SMN function in a tissue-specific fashion and therefore, address this issue directly. It was found that SMN is required in both neurons and muscle for normal NMJ morphology, since GAL4-inducible RNAi reduction of SMN in neurons and muscle both show a decrease in NMJ bouton numbers and GluRIIA staining. In addition, expression of SMN in either tissue is sufficient to partially rescue NMJ defects associated with loss of Smn function. These results are consistent with previous reports in Drosophila, zebrafish and mouse that indicate an interdependence of neuron and muscle SMN activity (Chang, 2008).
In contrast to a requirement for Smn in both muscle and neurons at the NMJ, muscle specific reduction of Smn causes a more severe lethal phenotype. The cause of the lethality is not known. It is possible that the earlier onset of lethality observed for the how24BGAL4 reduction of SMN may result from the leakiness of the driver or loss of SMN activity in dividing cells (the elavGAL4 driver expresses predominantly in post-mitotic cells). However, the results raise the possibility that the organism is more vulnerable to SMN reduction in the muscle. This is also consistent with the post-synaptic concentration of SMN at the NMJ. The functional relevance of these observations remains to be determined; however, a previous report has suggested that Smn may have a specific function in the Drosophila adult skeletal muscle where SMN is expressed in the sarcomere and was shown to bind to alpha-actinin (Rajendra, 2007). Together, these data provide plausible explanations why muscle may be rendered more susceptible to loss of Smn function (Chang, 2008).
Current therapeutic strategies for treatment of SMA are based on the dosage dependent nature of the disease, focusing on drugs that increase SMN2 transcription and splicing efficiency. Though these strategies may ultimately prove successful in treating SMA, complementary therapies may allow for the delivery of a combination of drugs as this has been shown to be successful in alleviating the symptoms of other diseases, such as AIDS. Hence, the identification and, ultimately, the manipulation of genetic elements that affect SMN activity may be necessary to treat SMA effectively. Though previous biochemical studies provide valuable and fundamental knowledge of SMN function, current understanding of SMN has been limited mainly to its binding partners and a few genetic modulators. Thus, a genome-wide genetic screen was performed in Drosophila to identify novel components of the Smn genetic circuitry to broaden knowledge of its function and to seek potentially novel therapeutic approaches beyond the augmentation of SMN2 expression (Chang, 2008).
Since the severity/onset of Smn-dependent mortality corresponds to the degree of NMJ defects, it was reasoned that the identification of enhancers and suppressors of Smn73Ao-dependent lethality would be likely to yield genes that also function at the NMJ. Genetic screen using an allele (Smn73Ao) that encodes a point mutation seen in SMA patients resulted in the identification of twenty-seven modifiers of Smn lethality. Though the genetic circuitry in Drosophila may differ from that which exists in humans, it is expected that there to be substantial overlap given the conservation of gene function across species (Chang, 2008).
Despite the essential role of SMN in snRNP assembly, an unexpected result of the screen was that none of the modifying insertions for which unambiguous gene assignments were made appear to function in RNA processing. Consistent with this notion, direct attempts to identify genetic relationships between SMN and known components of the SMN multimeric complex, including deficiencies that uncover the Drosophila Gemin homologs, did not affect the Smn73Ao heterozygous phenotype. One possible explanation is that removal of additional components of the SMN complex may not enhance Smn-related phenotypes since SMN activity is critical for the initial steps in SMN complex assembly. Hence, altering the activity of 'downstream' or directly-interacting partners of the SMN in the SMN complex may not affect Smn-related phenotypes (Chang, 2008).
Though none of the unambiguously identified modifier genes have an obvious role in snRNP assembly; genes were uncovered (wishful thinking, fmr1 and cutup) that have been shown function at the NMJ . Moreover, the majority of the remaining genes, which had no previously known NMJ function, also modified Smn NMJ phenotypes. Thus this genetic approach was efficient in identifying genes related to Smn NMJ function. This suggests that a similar approach utilizing a hypomorphic Smn allele (e.g. UAS-Smn-RNAi) that more closely approximates the dosage dependent nature of the human disease condition may identify additional members of the Smn genetic circuitry (Chang, 2008).
An analysis of the interacting loci according to molecular functions reveals an assortment of functional categories including cytoskeleton interaction proteins (moe and ctp), transcription factors (net) and metabolic enzymes (CG17323 and CG10561). Identified interactors also include members of several signal transduction pathways (e.g. BMP (wit), FGF (btl) and Nuclear Hormone Receptor (Eip75B)), raising the possibility that these evolutionarily conserved signaling pathways integrate with SMN or targets of SMN function(s). Though more detailed analyses of the nature of the links (synergistic or parallel) between these pathways and SMN are necessary, strong evidence is provided supporting a connection between BMP signaling and Smn at NMJ by testing additional upstream and downstream elements of this pathway. A molecular genetic analysis clearly indicates that SMN influences BMP activity. It remains to be determined whether SMN acts in the muscle to influence retrograde BMP signaling through the WIT receptor, for example by regulating the activity of the WIT ligand (GBB). It is also possible that SMN functions cell-autonomously in the neurons to affect the activity of MAD or its antagonist, DAD. Since the BMP signaling pathway has been implicated in other neurodegenerative diseases, including Duchenne Dystrophy and Marfan Syndrome, it is probable that BMP signaling also plays a role in the pathology of SMA in humans (Chang, 2008).
Similar to what is observed in SMA, the results confirm the susceptibility of the Drosophila NMJ to lower levels of SMN, and the screen has also identified several genes that modify Smn NMJ phenotypes. In other recent studies, micro-array based approaches analyzed the effect of reduced Smn levels on tissue-specific gene expression at a genome-wide level (Zhang, 2008; Lee, 2008). They identified genes whose splicing are susceptible to reduced SMN function (Zhang, 2008) and genes involved in general metabolic processes (Lee, 2008). These screens are clearly a valuable means to assess the housekeeping function of Smn. However, unlike the genes recovered from the current screen, most of which affect NMJ structure, it remains to be determined whether the genes identified through transcriptional profiling are involved in the development and/or maintenance of the NMJ. Thus, the genetic approach has uncovered elements, revealing a potential NMJ-specific role for Smn (Chang, 2008).
The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, no appreciable defects were found in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects (Praveen, 2012).
Transgenic expression of SmnWT was able to rescue the locomotion and viability defects observed in Smn null mutants, however, a majority of the snRNAs were largely unrestored to wild-type levels. Given the considerable degree of phenotypic rescue, this was an unexpected finding, suggesting that the observed reduction in snRNAs in the null animals is not a major contributor to larval locomotion and viability (Praveen, 2012).
Consistent with these findings, previous studies using mouse models of SMA have also reported decreases in snRNA levels, particularly for U12 and U4atac. Other studies have reported widespread pre-mRNA splicing changes in both minor- and major-class introns in late-symptomatic SMA mice. However, subsequent studies (Baumer, 2009) revealed that these splicing defects are likely a secondary consequence of severe SMN loss, as pre- and early-symptomatic SMA mice did not show an enrichment of unspliced introns (Praveen, 2012).
In the Drosophila system, it is noted that U12 and (to a lesser extent) U5 levels were partially rescued by transgenic expression of SmnWT and SmnT205I, although U4atac levels remained low. Thus, one explanation for the results could be that splicing of minor-class (U12-specific) introns is sensitive to small changes in snRNA concentrations and that U12 and U5 are limiting factors, whereas U4atac is not. This hypothesis was tested by using qRT-PCR to measure mRNA levels in 18 of the 20 predicted minor-class introns in the Drosophila transcriptome. A mutation in the U6atac gene resulted in pronounced defects in splicing of minor-class introns, however, splicing of these same mRNAs was largely unaffected in Smn null mutants. Among the minor-intron transcripts analyzed, the steady-state levels of two mRNAs, CG15081 (Phb2/Rea in mice) and CG33108 (ortholog unknown), were reduced by roughly 50% in Smn-/- larvae. It is possible that reduction in levels of one or more of these mRNAs could contribute to the phenotype of Smn null mutants, however, CG15081 heterozygotes are completely viable. A similar decrease in levels of the Phb2 mRNA in mice was shown to affect neither organismal viability nor motor function strongly suggesting that haploinsufficiency for CG15081 is not the cause of the Smn phenotype (Praveen, 2012).
These observations question the significance of snRNA levels in human SMA etiology. In this regard, it is important to note that recessive point mutations in the gene encoding human U4atac snRNA do not phenocopy SMA. Instead, loss of U4atac function causes a disease known as microcephalic osteodysplastic primordial dwarfism type I (MOPD I), which is characterized by severe intrauterine growth retardation and multiple organ abnormalities. Similar to the fruitfly U6atac mutants, cells derived from MOPD I patients display marked defects in splicing of minor-class introns, whereas splicing of major-class (U2-type) introns is unaffected. Although the possibility that tissue-specific defects in minor-intron splicing may be causative for SMA cannot be entirely excluded, the finding that transgenic expression of SmnWT can rescue viability and fertility without restoring U4atac levels effectively uncouples the observed global snRNA deficits from the organismal phenotype. Moreover, it is important to note that the Smn and U6atac mutants were analyzed just prior to onset of the lethal phase. At this timepoint, Smn null animals already display significant motor function defects. It is therefore concluded that perturbations in minor spliceosome levels are not likely to be causative for the larval locomotion and viability defects observed in Smn null mutants (Praveen, 2012).
With the possible exception of U5, SmnT205I animals have nearly identical snRNA profiles to those of the SmnWT animals, yet most of the T205I animals die as pupae. Given the intermediate phenotype of both humans and fruit flies expressing the T274I/T205I mutation and the fact that human SMNT274I is active in Sm-core assembly, these findings strongly suggest that mutation of this residue disrupts a second, essential function of SMN protein. This does not mean that splicing plays no role in downstream SMA pathology; it clearly does. There is strong evidence for a negative feedback loop wherein low levels of SMN protein exacerbate exon skipping of human SMN2, leading to a further reduction in SMN expression (Jodelka, 2010; Ruggiu, 2011). However, because Smn is a single-exon gene in Drosophila, this system system also uncouples protein-based defects in SMN from autologous feedback regulation via splicing. In conclusion, the results demonstrate that the reduction in snRNA levels observed in Smn mutants is not a major contributor to organismal lethality, and indicate that non-snRNP biogenesis functions of SMN play critical roles in the etiology of SMA. Molecular identification of this second SMN function will be an important subject of future investigation (Praveen, 2012).
Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. A Drosophila system has been developed to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle-specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with alpha-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity (Rajendra, 2007).
Spinal muscular atrophy (SMA) is a genetic disorder associated with recessive loss-of-function mutations in the human survival motor neurons 1 (SMN1) gene (Lefebvre, 1995). SMA is a broad-spectrum disorder whose severity is inversely proportional to levels of full-length SMN protein. The most severe form of SMA is also the most common one, and these patients typically die within the first 2 yr of life. SMA is characterized by loss of motoneurons from the anterior horn of the spinal cord and progressive muscular atrophy in the limbs and trunk, usually culminating in respiratory failure (Rajendra, 2007 and references therein).
SMN is the central member of a large oligomeric protein complex implicated in a variety of subcellular processes, including pre-mRNA transcription and splicing, RNP biogenesis and transport, neuritogenesis, and axonal pathfinding, as well as in the formation and function of neuromuscular junctions (Briese, 2005; Eggert, 2006). However, the only SMN function that has been well-documented to date is its role in the biogenesis of Sm-class small nuclear RNPs (snRNPs). Despite the observation that SMA patient-derived SMN1 mutations lead to defects in Sm-core assembly in vitro, a definitive link between snRNP biogenesis and the etiology of the disease has not been established in a model organism (Rajendra, 2007 and references therein)
Null mutations in single-copy SMN genes are lethal in every organism studied to date. In humans and higher primates, there are two SMN genes, SMN1 and SMN2. SMN2 is dispensable, but can partially compensate for homozygous loss of SMN1 (Monani, 2005). Patients with additional copies of SMN2 display milder phenotypes, a finding that has been confirmed using several transgenic mouse models (Monani, 2005). Because SMA is caused by reduced expression of SMN, modeling SMA in other genetically tractable organisms has been hampered by the need to create hypomorphic mutations. This study describes the generation of a Drosophila melanogaster model of SMA. Hypomorphic Smn mutants are characterized by an inability to fly or jump, and they display severe neuromuscular defects. The analysis of this phenotype has led to the surprising discovery that SMN is a sarcomeric protein, implicating a muscle-specific function (Rajendra, 2007).
Smn (CG16725) is a single-exon gene in Drosophila, encoding a 226-aa protein (Miguel-Aliaga, 2000). The expression profile shows that dSMN is highly expressed during embryogenesis, but that the levels decrease sharply during subsequent developmental stages. Because SMN is essential for Sm-core RNP assembly in human cells (Shpargel, 2005; Wan, 2005; Winkler, 2005), whether the Drosophila protein has a similar conserved function was investigated. Schneider 2 (S2) cells treated with double-stranded RNA (dsRNA) targeting Smn were efficiently and specifically depleted of dSMN. As assayed by two independent methods, Smn dsRNA-treated S2 cells were deficient in assembly of new Sm cores. Thus, it is concluded that SMN's function in snRNP assembly is conserved in invertebrates (Rajendra, 2007).
SMA is caused by reduced levels of SMN in mammals; complete loss of function results in early lethality (Monani, 2005). To generate a better Drosophila model for SMA, neuromuscular phenotypes were screened for in adult flies by imprecise excision of the P element in SmnE. From a total of ~170 independent excisions, two lines (SmnE2 and SmnE33) were isolated that displayed overt motor dysfunction. SmnE2 and SmnE33 homozygotes (henceforth referred to as E2 and E33 mutants, respectively) each showed marked defects in flying and jumping. The E2 mutants exhibited a 2-d delay in pupation, reflecting an extended larval period, and ~20% of the E2 pupae died at the pharate adult stage. However, the phenotype of the E2 mutants was incompletely penetrant; ~45% of E2 animals had flight and jump defects. Moreover, dSMN expression levels in these animals were also variable. In contrast, E33 mutants were completely viable and fertile, and 100% of the animals were incapable of flying or jumping. Because the E33 phenotype was fully penetrant, this allele was chosen for further characterization (Rajendra, 2007).
Despite the well-established gene-disease relationship between SMN1 and SMA, the connection between protein function and molecular etiology has been obscured by a plethora of putative cellular functions attributed to SMN. Previous investigations have shown that the SMN complex is required for assembly and transport of spliceosomal snRNPs. Additional findings point to roles for this complex in neurite outgrowth and pathfinding (Fan, 2002; McWhorter, 2003; Sharma, 2005), neuromuscular junction formation (Chan, 2003), profilin binding (Giesemann, 1999; Sharma, 2005), and axonal transport of ß-actin mRNPs (Rossoll, 2003). A common link between each of these additional studies is the actin cytoskeleton. The finding that reduced dSMN expression leads to motor axon routing and arborization defects, coupled with a loss of Act88F expression in the muscle, is consistent with this actin-related theme (Rajendra, 2007).
The vast majority of SMA studies continue to be focused on a motoneuron-specific role for SMN; the current results do not exclude such a function. However, the idea that SMN might also have a muscle-specific function is not a new one. Cocultures of SMA type I and II muscles with wild-type motoneurons failed to sustain innervation, whereas muscles from control or SMA type III patients maintained stable connections, suggesting a muscle-specific requirement for SMN. Similarly, down-regulation of Smn in mouse C2C12 cells revealed defects in myoblast fusion (Shafey, 2005) and tissue-specific knockouts of Smn in mouse muscle resulted in pronounced dystrophic phenotypes (Cifuentes-Diaz, 2001; Nicole, 2003). Also in support of a muscle-specific function is the observation that, despite having comparable levels of SMN, mouse skeletal muscle extracts failed to support efficient Sm-core assembly, whereas extracts from spinal cord were quite active. Collectively, these studies show that relatively high levels of SMN are required in muscles, although the reason for this requirement was unclear (Rajendra, 2007).
The discovery that SMN is a sarcomeric protein required for expression of muscle-specific actin not only provides a plausible role for the protein in muscles, but highlights the potential importance of this tissue in SMA pathophysiology. At least 20 different skeletal muscle diseases are thought to be caused by mutation or mislocalization of sarcomeric proteins. In this regard, it is particularly interesting that SMA patients have been shown to display varying degrees of myofibrillar/sarcomeric (including Z-line) abnormalities. Notably, αB-crystallin was recently reported to form a complex with SMN in HeLa cells (den Engelsman, 2005). αB-crystallin is an intermediate filament protein that, in muscle cells, accumulates at the Z-line. Thus, the SMN complex can interact with at least two distinct Z-line proteins, α-actinin, and αB-crystallin (Rajendra, 2007).
This study shows that reduced thoracic dSMN levels result in loss of Act88F expression with no apparent defect in either snRNP biogenesis or pre-mRNA splicing. Because expression of muscle-specific actin is known to be dependent on motoneuron innervation, the neuronal defects observed in the SmnE33 hypomorphs are consistent with those expected of an SMA model. Further, the data are consistent with denervation as either the cause or a consequence of muscle degeneration. Notably, a fraction of SMA type III patients display dystrophic phenotypes without evidence of neurogenic abnormalities. Although motoneuron loss is generally regarded as a late event in disease progression, one of the main problems in studying SMA, especially the severe forms, is that only the end-stage of the disease could be analyzed (Rajendra, 2007).
It is currently unknown whether the mutant phenotype observed in the Smn hypomorphs is caused by reduced dSMN expression in the thoracic muscles, the motoneurons, or a combination of tissues. Future work using tissue-specific rescue constructs and a detailed analysis of motoneuron development and myogenesis in the Smn hypomorphic pupae will address these important issues. Regardless of the actual disease trigger, the identification of SMN as a sarcomeric protein underscores the importance of muscle cell function in modulating the severity of SMA (Rajendra, 2007).
The C-terminal tails of spliceosomal Sm proteins contain symmetrical dimethylarginine (sDMA) residues in vivo. The precise function of this posttranslational modification in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and pre-mRNA splicing remains largely uncharacterized. This study examined the organismal and cellular consequences of loss of symmetric dimethylation of Sm proteins in Drosophila. Genetic disruption of dart5, also termed Capsuleen, the fly ortholog of human PRMT5, results in the complete loss of sDMA residues on spliceosomal Sm proteins. Similarly, valois, a previously characterized grandchildless gene, is also required for sDMA modification of Sm proteins. In the absence of dart5, snRNP biogenesis is surprisingly unaffected, and homozygous mutant animals are completely viable. Instead, Dart5 protein is required for maturation of spermatocytes in males and for germ-cell specification in females. Embryos laid by dart5 mutants fail to form pole cells, and Tudor localization is disrupted in stage 10 oocytes. Transgenic expression of Dart5 exclusively within the female germline rescues pole-cell formation, whereas ubiquitous expression rescues sDMA modification of Sm proteins and male sterility. This study has shown that Dart5-mediated methylation of Sm proteins is not essential for snRNP biogenesis. The results uncover a novel role for dart5 in specification of the germline and in spermatocyte maturation. Because disruption of both dart5 and valois causes the specific loss of sDMA-modified Sm proteins and studies in C. elegans show that Sm proteins are required for germ-granule localization, it is proposed that Sm protein methylation is a pivotal event in germ-cell development (Gonsalvez, 2006; full text of article).
Pre-messenger-RNA splicing, a hallmark feature of eukaryotic cells, is carried out by a large ribonucleoprotein (RNP) complex called the spliceosome. Numerous gene products are therefore dedicated to the task of building functional spliceosomes, the cellular machines that mediate the removal of intronic sequences. Small nuclear RNPs (snRNPs), central components of the spliceosome, are assembled in a highly orchestrated and sequential manner, involving maturation steps in both the nucleus and cytoplasm of the cell. The U1, U2, U4, and U5 spliceosomal snRNPs each contain a common set of seven core Sm proteins: SmB/B′, SmD1, SmD2, SmD3, SmE, SmF, and SmG. These proteins bind to a common sequence motif within the U snRNAs and form a heteroheptameric ring structure (Gonsalvez, 2006).
Assembly of the Sm ring takes place in the cytoplasm and, in vivo, requires the activity of the survival of motor neurons (SMN) protein complex. Mutations that reduce the level of SMN, the central member of this complex, result in a human neurogenetic disorder called spinal muscular atrophy (SMA). Importantly, cells from SMA patients display a reduced capacity for Sm core assembly. Collectively, the available data are consistent with the idea that SMA results from a general reduction in snRNP biogenesis, with motor neurons being particularly susceptible to reduced snRNP levels. However, the possibility that SMN functions in a novel cell-specific pathway has not been conclusively ruled out (Gonsalvez, 2006).
Three of the seven core Sm proteins, SmB/B′, SmD1, and SmD3, contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. The enzymes that catalyze this posttranslational modification are called protein arginine methyltransferases (PRMTs) and have been placed into two categories -- type I and type II. Type I enzymes mediate the more common modification, asymmetric dimethylarginine (aDMA). Type II enzymes are responsible for the less frequent sDMA modification. To date, the only known type II enzymes are PRMT5 and PRMT7, each of which is capable of methylating Sm proteins in vitro. Reduction of PRMT5 levels by RNA interference (RNAi) correlates with a decrease in the level of Sm-protein methylation in vivo. Furthermore, PRMT5 associates, along with MEP50/WD45 and pICln, in a complex that contains Sm proteins in vivo. Both MEP50 and pICln can directly bind to Sm proteins, thus making a strong case for involvement of the PRMT5 complex in Sm-protein methylation. It is not currently known whether PRMT7 plays any role in Sm-protein methylation, and binding partners for PRMT7 have not been described (Gonsalvez, 2006).
The precise role of Sm-protein methylation in snRNP biogenesis remains a poorly understood topic. In vitro, SMN protein preferentially binds to C-terminal peptides, derived from SmD1 and SmD3, that contain sDMA but not aDMA residues. The prevailing view holds that sDMA modification of Sm proteins serves to recruit SMN, thus facilitating efficient transfer of Sm proteins from the PRMT5 complex to the SMN complex for assembly of the Sm core. A prediction that follows from this interpretation is that symmetric dimethylation of Sm proteins is a requirement for efficient snRNP biogenesis. This hypothesis was explored in vivo, with Drosophila melanogaster. For these experiments, a fly strain containing an insertion in the dart5 gene, the fly ortholog of human PRMT5, was used. Lysates prepared from homozygous mutant flies display a complete and specific loss of sDMA modification of Sm proteins. Surprisingly, homozygous disruption of dart5 is not lethal, and the expected number of progeny is recovered. Using additional molecular assays, it was found that spliceosomal snRNP biogenesis was similarly unaffected. Instead, it was found that dart5 males were completely sterile, with defects in spermatogenesis. In contrast to the males, the homozygous mutant females were fertile. However, the progeny obtained from homozygous dart5 mothers were sterile and agametic. Consistent with this finding, embryos from dart5 females were devoid of pole cells, the germline precursors. This is reminiscent of the classic 'grandchildless' phenotype described for a number of genes such as tudor, vasa, and valois. Interestingly, it was recently shown that valois is the Drosophila ortholog of human MEP50/WD45. Like their human counterparts PRMT5 and MEP50, the Valois and Dart5 (also known as Capsuléen) proteins were recently shown to associate in the fly. Plausibly, these two gene products may function in a related and perhaps overlapping pathway that contributes to germ-cell specification. Notably, it was found that, similar to the valois mutant phenotype, Tudor protein was mislocalized in dart5 mutant ovaries. On the basis of these and other findings, it is proposed that sDMA modification of Sm proteins represents a critical step in the specification and maintenance of the germ-cell lineage (Gonsalvez, 2006).
Although Dart5 activity is required for Tudor function, dart5 does not fit the mold of a classical posterior-group gene. In order to be placed directly in the germ-cell specification pathway, upstream of tudor, the dart5 phenotype should be at least as strong as that of tudor. It is not. Similarly, mutations in vasa do not have an appreciable effect on Dart5 activity, as measured by Sm-protein methylation. Thus a revised model of the germ-cell specification pathway is proposed, wherein dart5 (and valois) primarily affect Tudor localization, resulting in a loss of pole-cell formation. However, unlike oskar and vasa mutations, somatic patterning appears to be relatively unaffected. Because 15% of tudor null embryos develop normally, it has been suggested that Tudor is not directly required for posterior patterning. However, tudor null embryos contain fewer polar granules than wild-type embryos and never form pole cells. Thus a fully functional pole plasm may be required for stabilizing the level or maintaining the localization of factors involved in establishing the body plan. In such a scenario, it is not surprising that a subset of tudor null embryos display patterning defects. Given that mutation of dart5 appears to compromise Tudor function, a small fraction of dart5-1 embryos also display patterning defects (Gonsalvez, 2006).
The elevated hatching frequency of dart5-1 as compared to tudor null embryos and the residual accumulation of Oskar in dart5-1 blastoderm embryos suggest that a partially functional pole plasm is formed in the absence of Dart5. However, this level of functionality is insufficient to mediate germ-cell specification, given that 100% of the embryos that develop are agametic. Because Tudor is only modestly reduced in dart5-1 mutant ovaries in comparison to its complete absence from tudor null ovaries, it is logical that the dart5-1 phenotype would be less severe than the tudor null phenotype. Although Tudor was not enriched at the posterior pole in dart5-1 oocytes, neither was it completely absent from this location. As such, the residual level of Tudor, along with properly localized Oskar and Vasa, might be sufficient to assemble a partially functional pole plasm in the oocyte (Gonsalvez, 2006).
Given their in vivo association, it is not surprising that valois and dart5 share many phenotypes: absence of pole cells, male sterility, and loss of Sm-protein sDMA residues. Despite the similarity of the mutant phenotypes, there are a few differences worth noting. For instance, the spermatocyte maturation defect was less severe in the valois mutant as compared to dart5-1. Additionally, unlike the dart5-1 mutant, the vls3 mutant displayed a rather strong maternal-effect lethal phenotype. This result is consistent with a previous finding that valois mutants displayed pleiotropic defects during cellularization. In addition, whereas valois mutants affect the level of Oskar protein in ovaries, there is no apparent Oskar defect in dart5-1 mutants. Thus Valois may have additional functions outside of its complex with Dart5 (Gonsalvez, 2006).
This report has identified Sm proteins as in vivo targets of Dart5. Furthermore, it was shown that Valois is also required for the sDMA modification of Sm proteins and proper expression of Dart5. In the absence of Dart5 and Valois, germ-cell specification, but not general snRNP biogenesis, is disrupted. These observations point to a model whereby Sm proteins, or more precisely symmetrical arginine dimethylation of Sm proteins, play a critical role in germ-cell specification. Consistent with this hypothesis, Sm proteins are thought to play a specific role, unrelated to splicing, in P granule integrity germ-cell specification in C. elegans. P granules are structurally and functionally related to the nuage of Drosophila. Like the Drosophila nuage, P granules are RNA rich and contain a number of proteins that have critical roles in germ-cell development. Importantly, Valois is localized to, and is required for, the proper formation of the nurse-cell nuage in Drosophila. Another prominent component of the Drosophila nuage is Tudor. In mouse spermatocytes, Mouse-Tudor-Repeat gene1 (MTR-1) is localized to the nuage and specifically associates with Sm proteins therein. Furthermore, the nuage of Xenopus oocytes was also shown to specifically contain Sm proteins. It will therefore be of great interest to determine whether Sm proteins are components of the nuage in Drosophila. In the absence of Dart5 activity, prominent Tudor localization to the nuage is disrupted. Sm-protein methylation may therefore be required for maintaining proper integrity of the Drosophila nuage. In order to more fully explore this hypothesis, ultrastructural analyses will be required (Gonsalvez, 2006).
Tudor is the founding member of a family of proteins that contain Tudor domains. Several lines of evidence point to a function for Tudor domains as methyl binding protein modules: (1) the SMN protein contains a single Tudor domain, mutation of which causes a significant decrease in binding affinity for Sm proteins; (2) molecular modeling studies suggest that Tudor domains are structurally related to other domains, such as the Chromo domain, that are known to bind methylated proteins; (3) SMN binding to Sm proteins decreases upon loss of methylation; (4) it has been shown that two other Tudor-domain proteins, splicing factor 30 (SPF30) and Tudor-domain-containing 3 (TDRD3) interact with Sm proteins in a methylation-dependent manner. Drosophila Tudor contains 11 such protein motifs. Thus, it is plausible that Tudor interacts with sDMA residues within the C termini of Sm proteins and that this interaction is somehow required for Tudor function and, consequently, for germ-cell development. Experiments designed to examine this hypothesis are ongoing. In this regard, it is noteworthy that disruption of dart5 affects the levels of Tudor protein and its localization within the egg chamber (Gonsalvez, 2006).
This study has shown that symmetrical dimethylation of arginine residues within the Sm proteins is lost upon disruption of dart5, the Drosophila ortholog of PRMT5. The possibility cannot be ruled out that, in the absence of Dart5 activity, Sm proteins might contain other posttranslational modifications (e.g., monomethylated or asymmetrically dimethylated arginine residues). However, correlated with the loss of symmetric dimethylation of Sm proteins is a complete failure to develop germ cells in subsequent generations. Expression of myc-tagged Dart5 only in the female germline via a nanos driver rescued pole-cell formation in early embryos and Vasa localization to the developing gonad. One interpretation of these observations is that symmetric dimethylation of Sm proteins plays a central role in specifying the germline. The dart5-1 allele will be a valuable tool in exploring this hypothesis. If Sm proteins do play a role in germ-cell specification, simple mutational or knockout experiments will not be useful in uncovering the mechanism, because these alterations cause somatic-cell lethality. RNAi of Sm proteins in C. elegans, while causing a disruption in the localization and integrity of P granules, is also coupled with embryonic lethality. The available mutations in Drosophila Sm proteins are likewise all lethal (Gonsalvez, 2006).
These results suggest that Sm proteins play at least two distinct roles in the organism, one a general function in pre-mRNA splicing and the other in germ-cell specification and maintenance. The dart5-1 allele is very informative in this regard because it uncouples these two functions: snRNP biogenesis and splicing are ongoing in dart5-1 homozygotes, but germ-cell specification is disrupted. Given the similar phenotypes of the dart5 and valois mutants, the function of the Tudor domain, the delocalization of Tudor in dart5-1 egg chambers, and the available data on the localization of Sm proteins to the nuage in several different species, the strongest interpretation favors a critical role for Sm proteins in germ-cell specification. Although this hypothesis is favored, it is not possible to rule out the possibility that, for example, loss of methylation of some other protein causes the observed phenotypes. Future work should provide much-needed mechanistic insight into this question. In this regard, it will be particularly important to determine whether Sm proteins are components of the nuage and pole plasm in Drosophila. If so, it will be most interesting to elucidate whether they are associated with snRNAs or are complexed with a different class of RNA (Gonsalvez, 2006).
Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, this topic was examined in detail and it was found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, it was found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, a fly strain was constructed that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. It is therefore concluded that dart5 is required for viability when SMN is limiting (Gonsalvez, 2008).
The survival motor neuron (SMN) protein, the determining factor for spinal muscular atrophy (SMA), is complexed with a group of proteins in human cells. Gemin3 is the only RNA helicase in the SMN complex. This study reports the identification of Drosophila Gemin3 and investigates its function in vivo. Like in vertebrates, Gemin3 physically interacts with SMN in Drosophila. Loss of function of gemin3 results in lethality at larval and/or prepupal stages. Before they die, gemin3 mutant larvae exhibit declined mobility and expanded neuromuscular junctions. Expression of a dominant-negative transgene and knockdown of Gemin3 in mesoderm cause lethality. A less severe Gemin3 disruption in developing muscles leads to flightless adults and flight muscle degeneration. These findings suggest that Drosophila Gemin3 is required for larval development and motor function (Cauchi, 2008).
Gemin3 or DP103 was first identified in mammalian culture cells through biochemical approaches. The Gemin3 protein has three critical features. First, the N-terminus of Gemin3 contains multiple helicase motifs including a DEAD-box. Second, Gemin3 interacts with SMN in vitro and in vivo (Otter, 2007). Third, the Gemin3 and SMN proteins have a similar subcellular localization pattern (Charroux, 1999; Zhang, 2006; Cauchi 2008 and references therein).
In Drosophila there are 29 DEAD-box RNA helicases. Using human and mouse Gemin3 to BLAST the Drosophila genome, CG6539, previously identified as DEAD/DEAH RNA helicase 1 (Dhh1), is the top hit. In the N-terminus, CG6539 contains 9 conserved RNA helicase motifs including a DEAD-box. A segment in the middle of CG6539, which corresponds to the SMN-binding domain in human Gemin3, is less conserved. Moreover, co-immunoprecipitation experiments using Drosophila larval muscle extracts show that Gemin3 binds to SMN in vivo. Localization assays demonstrate that Gemin3 co-localizes with SMN in the cytoplasm and nucleus. Taken together, this study has identified the Drosophila orthologue of vertebrate Gemin3 (Cauchi, 2008).
Recently, an independent study by Kroiss (2008) also identified CG6539 as Drosophila Gemin3 through bioinformatic and biochemical approaches using Drosophila culture cells. Both their study in Drosophila culture cells and this study in Drosophila tissues have shown that Gemin3 interacts with SMN, suggesting that Gemin3 is a bona fide component of the SMN complex in fruit flies, similar to that in vertebrate systems (Cauchi, 2008).
Multiple lines of evidence are presented demonstrating that Drosophila Gemin3 is essential for animal development and survival. (1) Homozygous loss of gemin3 through a specific transposon insert (gemin3R) or a transheterozygous combination of two transposon inserts which do not complement each other (gemin3R/gemin3W) results in lethality at the larval and/or prepupal stage. (2) A functional gemin3 transgene specifically rescues the lethality and developmental defects caused by loss of gemin3. (3) Expression of a dominant-negative allele of gemin3 (gemin3?N) or Gemin3 knockdown by RNAi ubiquitously or even in a tissue-specific pattern results in lethality or reduced viability (Cauchi, 2008).
Gemin3-null mutants have recently been described in the mouse. Heterozygous gemin3 mutant mice are healthy and fertile, with minor defects in the female reproductive system, whereas homozygous gemin3 knockout in mice leads to death at the 2-cell embryonic stage (Mouillet, 2008). Thus, the lethality caused by loss of Gemin3 in Drosophila is consistent with the findings in Gemin3-null mice. However, while Gemin3-null mice died at an early embryonic stage, gemin3 mutant flies exhibit delayed lethality, which probably results from maternal contribution of the gemin3 transcript. In a separate study in female ovaries, severe defects were observed in nurse cells and oocytes when gemin3 is disrupted in germline cells (Cauchi, 2008).
The earliest clues pointing towards a motor function were a progressive loss of mobility and consequent long and thin puparia when Gemin3 function is lost. Similar phenotypes have previously been observed in mutants with disrupted Mlp84B, a muscle sarcomeric protein, or Tiggrin, an extracellular matrix ligand for the PS2 integrins. gemin3 mutants have an overgrown NMJ though these could be a secondary response to the progressive loss of muscle power. The size ratio of NMJs to muscles is reduced when gemin3 is overexpressed raising the possibility that Gemin3 might also play a role in synaptic growth (Cauchi, 2008).
The requirement of Gemin3 in mesoderm and larval muscles for adult viability suggests a function of Gemin3 at the post-synaptic side. Based on the tissue-specific phenotypes uncovered, such a function is critical for pupal metamorphic changes and flight muscles. However, another possible explanation is that an earlier and wider disruption of Gemin3 by mesodermal-related drivers is responsible for the lethality, while late and local disruption of Gemin3 by neuroectodermal-related drivers causes milder phenotypes. More studies on the expression details of Gemin3 in pre- and post-synaptic tissues would help to distinguish those views (Cauchi, 2008).
Studies in vertebrate systems have shown that Gemin3 directly binds to SMN (Otter, 2007). A recent study in Drosophila culture cells (Kroiss, 2008) and this study in fly tissues confirm that the interaction between Gemin3 and SMN is conserved from fly to human (Cauchi, 2008).
This study raises the possibility of a functional interaction between Gemin3 and SMN. Loss of gemin3 phenocopies the larval mobility phenotypes observed in smn mutants (Chan, 2003). Strong Gemin3 disruption in mesoderm and muscles led to striking developmental defects during metamorphosis, similar to those reported on disruption of SMN in a similar expression pattern (Miguel-Aliaga, 2000). A less severe gemin3 disruption in the developing musculature results in viable but flightless adult flies, which have flight muscle degeneration, similar to the phenotype in a hypomorphic smn mutant (Cauchi, 2008).
This study observed that gemin3 mutants exhibit an overgrown NMJ before puparation and overexpression of gemin3 leads to a significant decrease in NMJ area and branches relative to muscle size. Interestingly, two studies describe a range of NMJ phenotypes for smn mutants (Chan, 2003; Chang, 2008). It is still not clear whether smn and gemin3 mutants have similar morphologic defects at the NMJ as the parameters and the segments used for NMJ analysis vary in different studies. Comparison of smn and gemin3 mutant NMJs with the same standard, as well as analysing the NMJ phenotype in smn and gemin3 double mutants would help to address this question (Cauchi, 2008).
The motor defects unravelled on disruption of Gemin3 function in Drosophila are very intriguing in view of its association with SMN, and the possible link to SMA. More studies are necessary to clarify the roles of SMN-Gemin3 interaction in development, which may help in the understanding of the molecular mechanisms of the devastating neurodegenerative disorder SMA (Cauchi, 2008).
The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. This report identifies and characterizes the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with Drosophila SMN in vitro and in vivo. RNA interference for Gem3 codepletes SMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete SMN. Transgenic overexpression of Gem3 rescues lethality, but overexpression of SMN does not, indicating that loss of SMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. These results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction (Shpargel, 2009).
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease with a carrier frequency of 1 in 50 unrelated individuals and is distinguished by degeneration of spinal motor neurons and severe atrophy of skeletal muscle. The Survival Motor Neuron 1 gene (SMN1) was identified by positional cloning as the gene responsible for ~95% of SMA cases. Because of the observed variability in phenotypic severity, at least three classes of SMA have been established. SMA type I, also known as Werdnig-Hoffman disease, is the most common and the most severe form of the disease, with an age of onset at <6 mo. SMA type I patients do not survive and typically die within the first 24 mo. SMA type II is an intermediate form, with an age of onset in the first 18 mo, and these patients often survive well into their teens. SMA type III, or Kugelberg-Welander syndrome, is characterized by late onset (after 18 mo) and chronic muscle weakness without a significant decrease in lifespan. All three classes of SMA are allelic, caused by mutations in SMN1 (Shpargel, 2009 and references therein).
Interestingly, the human genome contains a second locus, SMN2, which produces reduced amounts of full-length SMN protein and cannot fully compensate for the loss of SMN1. Complete loss of Smn function results in early embryonic lethality in mice; animals that carry low-copy SMN2 transgenes survive embryogenesis but die postnatally, yet those with high-copy transgenes are completely viable. Thus, SMA can be viewed as a 'protein-dosage' disease, an interpretation that correlates well with the fact that SMA severity is inversely proportional to SMN protein levels (Shpargel, 2009 and references therein).
SMN is part of a large, oligomeric protein complex that is essential for a number of distinct steps in the biogenesis of metazoan Sm-class small nuclear ribonucleoproteins. SMN localizes diffusely throughout the cytoplasm, with intense nuclear signals corresponding to Cajal bodies (Liu, 1996; Matera, 1998). Based on the known protein-protein interactions, organization of the complex centers around SMN, which directly interacts with itself, Gemin2, Gemin3, Gemin5, and Gemin8. Gemin8 is thought to recruit Gemin6, Gemin7, and unr-interacting protein (UNRIP/STRAP), whereas Gemin3 brings Gemin4 into the complex. The SMN complex binds directly to the snRNA and to Sm proteins in order to coordinate snRNP assembly. It has been demonstrated by RNA interference (RNAi) knockdown that SMN, Gemin2, Gemin3, and Gemin4 are each required for efficient snRNP assembly in HeLa cells (Shpargel, 2005). Current theories suggest that Gemins and associated proteins function together to mediate the various steps of snRNP biogenesis. However, despite the excellent correlation between SMN protein levels and disease phenotype, mutations in other members of the SMN complex have not been associated with human disease (Shpargel, 2009 and references therein).
Genetic analysis in model organisms provides a unique opportunity to study factors contributing to disease pathogenesis. Drosophila SMN (dSMN) has been identified on the basis of sequence and functional conservation, and null mutations within the gene are larval lethal in the second and third instar stages (Chan, 2003; Rajendra, 2007). These larvae exhibit motor and neuromuscular defects. An adult model for Drosophila SMA was also generated. A hypomorphic mutation, called SmnE33, was created by imprecise excision of a P-element residing in the upstream control region (Rajendra, 2007). SmnE33 homozygotes exhibit reduced dSMN protein levels in the thorax of the adult fly. This deficiency leads to severe neuromuscular defects, including flightlessness, all of which can be rescued by expression of a YFP-Smn transgene (Rajendra, 2007). Notably, SMN is a sarcomeric protein in both flies and mice, and because snRNPs are absent from myofibrils, SMN likely performs a tissue-specific function in muscle (Rajendra, 2007). Other members of the Drosophila SMN complex have not been described (Shpargel, 2009).
This study identified and characterized Gemin3 (Gem3) as a member of the Drosophila SMN complex. Like its human counterpart, Gem3 interacts directly with SMN in vitro and in vivo. Furthermore, these two proteins colocalize in the Drosophila Cajal body and are required for efficient assembly of Sm snRNPs. Previously uncharacterized transposon insertions in Gemin3 and Smn exhibit larval motor defects, developmental delay, and a failure to pupate. The current results demonstrate the conservation of Gemin3 function in the fruitfly SMN complex and establish its essential role in various aspects of Drosophila development (Shpargel, 2009).
Gem3 and SMN colocalize to Drosophila Cajal bodies and are required for efficient Sm core snRNP assembly in S2 cells. In human cells, Gemin3 interacts strongly with Gemin4 and forms a subcomplex with this protein. Database searches have failed to identify putative Gemin4 orthologues in nonvertebrate species (Kroiss, 2008). Similarly, Gemins6-8, form distinct subcomplexes in human cells, but orthologues of these proteins have not been identified in the Drosophila genome (Kroiss, 2008). With the possible exception of Gemin2, budding yeast genomes do not appear to contain any of the known SMN complex proteins. Fission yeast, however, encode clear Smn and Gemin2 orthologues. Despite these facts, there is little evidence for a role for Smn in snRNP assembly in Schizosaccharomyces pombe or even for a cytoplasmic phase for Sm core assembly in Saccharomyces cerevisiae. These and other findings suggest that Drosophila contains a primitive version of the mammalian SMN complex (Shpargel, 2009 and references therein).
Although database searches suggest that many of the SMN complex proteins are not conserved in the fly, putative orthologues of Gemin2 (CG10419), Gemin5 (rigor mortis; CG30149), and UNRIP/STRAP (wmd; CG3957) can be identified. Several lines of evidence suggest that these proteins function together. Endogenous Drosophila SMN copurifies with Flag-Gemin3 and Flag-Gemin2. Kroiss (2008) also reported that SMN forms complexes with Gem3 in S2 cells. However, Gem3 appears to be weakly or transiently associated with SMN, as this protein was not recovered when Flag-SMN or Flag-Gemin2 were used for the purification pulldowns. Thus it is possible that Gem3 is present in substoichiometric amounts relative to SMN and Gem2. Despite the relative dearth of biochemical purification data linking these three factors into a single complex, this study found that Gem3 is required for Sm core assembly in vitro. Moreover, RNAi knockdown of Gem3 in S2 cells and transposon insertions in the Gemin3 locus in vivo resulted in codepletion of SMN. Importantly, overexpression of YFP-Smn in the Gemin3 null mutant background failed to rescue the lethality. Thus, although dGem3 may function to stabilize SMN, it may have a separate function inside or outside of the SMN complex. Additional experiments will be required to distinguish among these possibilities (Shpargel, 2009).
Evidence supporting a role for the WD-repeat protein Rigor mortis (rig/dGem5) in SMN complex function comes from phenotypic analyses. rigor mortis is an essential gene, and mutants therein display significant larval lethality; animals that escape the initial wave of larval lethality are developmentally delayed and fail to pupate (Gates, 2004). These phenotypes are strikingly similar to those of the SmnF and Gemin3 alleles described in this work. Gates have shown that rig/dGem5 interacts with several members of the ecdysone signaling pathway required for initiation of puparium formation. Mammalian Gemin5 is also involved in signal transduction (Kim, 2007). Similarly, UNRIP/STRAP, another WD repeat protein is an exclusively cytoplasmic member of the SMN complex (Carissimi, 2005; Grimmler, 2005) and is involved in intracellular signaling (Datta, 1998; Datta, 2000; Anumanthan, 2006). In the future, it will be interesting to determine whether rigor mortis interacts genetically and physically with other members of the Drosophila SMN complex (Shpargel, 2009).
Irrespective of potential roles for the SMN complex in signal transduction, the current results demonstrate the essential role that Gemin3 plays in organismal development. During manuscript revision of this article, Mouillet (2008) showed that the murine ortholog of Gemin3 (Dp103/Ddx20) is essential for early embryonic development in mammals. Loss-of-function mutations in Gemin3 have not been described in other organisms. To date, several Smn and Gemin2 alleles have been characterized. Null mutations in mouse Smn and Gemin2 are also early embryonic lethals (Schrank, 1997; Jablonka, 2002). Expression of a low-copy human SMN2 transgene rescues the embryonic lethality, but the mice die shortly after birth and display severe motor neuron degeneration and muscular atrophy phenotypes. Depletion of Smn in zebrafish embryos by morpholino injection elicits defects in motor axon outgrowth, although the primary versus secondary nature of the reported Smn phenotypes is unclear and the results seem to depend on the extent of depletion. Interestingly, depletion of Gemin2 is reported to have conflicting effects on motor axon development, possibly because of differences in the levels of gene inhibition or in the methods of phenotypic analysis (Shpargel, 2009 and references therein).
The connection between snRNP biogenesis and SMA is certainly complicated and is not well understood. This study has shown that mutation of two members of the Drosophila SMN complex, Smn and Gemin3, causes defects in larval motor function. In addition to larval Smn mutants, this laboratory has previously reported SMA-like phenotypes in adult flies containing a hypomorphic SmnE33 mutation (Rajendra, 2007). Thus, although it is clear that perturbations in the SMN complex can indeed result in neuromuscular dysfunction, the contribution that snRNP biogenesis plays in the etiology of these phenotypes remains a subject of ongoing investigation (Shpargel, 2005; Wan, 2005; Winkler, 2005; Gabanella, 2007). Further complicating interpretation of the various SMA models is the fact that the SMN complex appears to function in tissue-specific pathways involved in both neuronal and muscular development. Clearly, animal models will play an important role in future research aimed at distinguishing among the various functions of the SMN complex (Shpargel, 2009).
Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases this study confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. Use was made of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport (Cauchi, 2010).
Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. This study has used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, the conservation of modifier gene function was tested across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species (Dimitriadi, 2010).
Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. A Drosophila smn mutant has been isolated. The fly alleles contain point mutations in smn similar to those found in SMA patients. This study shows that aygotic smn mutant animals show abnormal motor behavior, and smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects (Chan, 2003; Full text of article)
The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. This study determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, widespread perturbations were observed in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1; see Drosophila Uba1). SMN physically interacts with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways lead to beta-catenin accumulation, and pharmacological inhibition of beta-catenin robustly ameliorates neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of beta-catenin is restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of beta-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. These data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent beta-catenin signaling, highlighting ubiquitin homeostasis and beta-catenin as potential therapeutic targets for SMA (Wishart, 2O14).
Survival motor neuron (SMN) is the determining factor in spinal muscular atrophy, the most common genetic cause of childhood mortality. SMN regulates stem cell division, proliferation and differentiation in Drosophila. However, it is unknown whether a similar effect exists in vertebrates. This study shows that SMN is enriched in highly proliferative embryonic stem cells (ESCs) in mice and reduction of SMN impairs the pluripotency of ESCs. Moreover, SMN reduction was found to activate ERK signaling and affects neuronal differentiation in vitro. Teratomas with reduced SMN grow more slowly and show weaker signals of neuronal differentiation than those with a normal level of SMN. Finally, over-expression of SMN was shown to be protective for ESCs from retinoic acid-induced differentiation. Taken together, these results suggest that SMN plays a role in the maintenance of pluripotent ESCs and neuronal differentiation in mice (Chang, 2014).
In spinal muscular atrophy, a neurodegenerative disease caused by ubiquitous deficiency in the survival motor neuron (SMN) protein, sensory-motor synaptic dysfunction and increased excitability precede motor neuron (MN) loss. Whether central synaptic dysfunction and MN hyperexcitability are cell-autonomous events or they contribute to MN death is unknown. These issues were addressed using a stem-cell-based model of the motor circuit consisting of MNs and both excitatory and inhibitory interneurons (INs) in which SMN protein levels are selectively depleted. SMN deficiency was shown to induce selective MN death through cell-autonomous mechanisms, while hyperexcitability is a non-cell-autonomous response of MNs to defects in pre-motor INs, leading to loss of glutamatergic synapses and reduced excitation. Findings from this in vitro model suggest that dysfunction and loss of MNs result from differential effects of SMN deficiency in distinct neurons of the motor circuit and that hyperexcitability does not trigger MN death (Simon, 2016).
Search PubMed for articles about Drosophila Smn
Anumanthan, G., Halder, S. K., Friedman, D. B. and Datta, P. K. (2006). Oncogenic serine-threonine kinase receptor-associated protein modulates the function of Ewing sarcoma protein through a novel mechanism. Cancer Res 66: 10824-10832. PubMed ID: 17108118
Battle, D. J., Kasim, M., Wang, J. and Dreyfuss, G. (2007). SMN-independent subunits of the SMN complex. Identification of a small nuclear ribonucleoprotein assembly intermediate. J Biol Chem 282: 27953-27959. PubMed ID: 17640873
Baumer, D., Lee, S., Nicholson, G., Davies, J. L., Parkinson, N. J., Murray, L. M., Gillingwater, T. H., Ansorge, O., Davies, K. E. and Talbot, K. (2009). Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy. PLoS Genet 5: e1000773. PubMed ID: 20019802
Borg, R. and Cauchi, R. J. (2013). The gemin associates of survival motor neuron are required for motor function in Drosophila. PLoS One 8: e83878. PubMed ID: 24391840
Briese, M., Esmaeili, B. and Sattelle, D. B. (2005). Is spinal muscular atrophy the result of defects in motor neuron processes? Bioessays 27: 946-957. PubMed ID: 16108074
Carissimi, C., Baccon, J., Straccia, M., Chiarella, P., Maiolica, A., Sawyer, A., Rappsilber, J. and Pellizzoni, L. (2005). Unrip is a component of SMN complexes active in snRNP assembly. FEBS Lett 579: 2348-2354. PubMed ID: 15848170
Carissimi, C., Saieva, L., Baccon, J., Chiarella, P., Maiolica, A., Sawyer, A., Rappsilber, J. and Pellizzoni, L. (2006). Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly. J Biol Chem 281: 8126-8134. PubMed ID: 16434402
Cauchi, R. J., Davies, K. E. and Liu, J.-L. (2008). A Motor Function for the DEAD-Box RNA Helicase, Gemin3, in Drosophila. PLoS Genet 4(11): e1000265. PubMed ID: 19023405
Cauchi, R. J., Sanchez-Pulido, L. and Liu, J. L. (2010). Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies. Exp Cell Res 316: 2354-2364. PubMed ID: 20452345
Cauchi, R. J. (2012). Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis. BMC Res Notes 5: 120. PubMed ID: 22361416
Chan, Y. B., et al. (2003). Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum. Mol. Genet. 12: 1367-1376. PubMed ID: 12783845
Chang, H. C., et al. (2008). Modeling spinal muscular atrophy in Drosophila. PLoS ONE 3: e3209. PubMed ID: 18791638
Chang, W. F., Xu, J., Chang, C. C., Yang, S. H., Li, H. Y., Hsieh-Li, H. M., Tsai, M. H., Wu, S. C., Cheng, W. T., Liu, J. L. and Sung, L. Y. (2014). SMN is required for the maintenance of embryonic stem cells and neuronal differentiation in mice. Brain Struct Funct [Epub ahead of print]. PubMed ID: 24633826
Chari, A., Golas, M. M., Klingenhager, M., Neuenkirchen, N., Sander, B., Englbrecht, C., Sickmann, A., Stark, H. and Fischer, U. (2008). An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs. Cell 135: 497-509. PubMed ID: 18984161
Charroux, B., et al. (1999). Gemin3: a novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of Gems. J. Cell Biol. 147: 1181-1194. PubMed ID: 10601333
Cifuentes-Diaz, C., et al. (2001). Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J. Cell Biol. 152: 1107-1114. PubMed ID: 11238465
Datta, P. K., Chytil, A., Gorska, A. E. and Moses, H. L. (1998). Identification of STRAP, a novel WD domain protein in transforming growth factor-beta signaling. J. Biol. Chem 273: 34671-34674. PubMed ID: 9856985
Datta, P. K. and Moses, H. L. (2000). STRAP and Smad7 synergize in the inhibition of transforming growth factor beta signaling. Mol. Cell. Biol 20: 3157-3167. PubMed ID: 10757800
den Engelsman, J., et al. (2005). Nuclear import of αB-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G. J. Biol. Chem. 280: 37139-37148. PubMed ID: 16129694
Dimitriadi, M., Sleigh, J. N., Walker, A., Chang, H. C., Sen, A., Kalloo, G., Harris, J., Barsby, T., Walsh, M. B., Satterlee, J. S., Li, C., Van Vactor, D., Artavanis-Tsakonas, S. and Hart, A. C. (2010). Conserved genes act as modifiers of invertebrate SMN loss of function defects. PLoS Genet 6: e1001172. PubMed ID: 21124729
Eggert, C., Chari, A., Laggerbauer, B. and Fischer, U. (2006). Spinal muscular atrophy: the RNP connection. Trends Mol. Med. 12: 113-121. PubMed ID: 16473550
Fan, L. and Simard, L. R. (2002). Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development. Hum. Mol. Genet. 11: 1605-1614. PubMed ID: 12075005
Fischer, U., Liu, Q. and Dreyfuss, G. (1997). The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell 90: 1023-1029. PubMed ID: 9323130
Gabanella, F., Butchbach, M. E., Saieva, L., Carissimi, C., Burghes, A. H. and Pellizzoni, L. (2007). Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs. PLoS ONE 2: e921. PubMed ID: 17895963
Garcia, E. L., Lu, Z., Meers, M. P., Praveen, K., Matera, A. G. (2013) Developmental arrest of Drosophila survival motor neuron (Smn) mutants accounts for differences in expression of minor intron-containing genes. RNA. [Epub ahead of print] PubMed ID: 24006466
Gates, J., Lam, G., Ortiz, J. A., Losson, R. and Thummel, C. S. (2004). rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development. Development 131: 25-36. PubMed ID: 14645129
Giesemann, T., et al. (1999). A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems. J. Biol. Chem. 274: 37908-37914. PubMed ID: 10608857
Gonsalvez, G. B., Rajendra, T. K., Tian, L. and Matera, A. G. (2006). The Sm-protein methyl transferase, dart5, is essential for germ-cell specification and maintenance. Curr. Biol. 16: 1077-1089. PubMed ID: 16753561
Gonsalvez, G. B., Praveen, K., Hicks, A. J., Tian, L. and Matera, A. G. (2008). Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster. RNA 14(5): 878-87. PubMed ID: 18369183
Grice, S. J. and Liu, J. L. (2011). Survival motor neuron protein regulates stem cell division, proliferation, and differentiation in Drosophila. PLoS Genet 7: e1002030. PubMed ID: 21490958
Grimmler, M., Otter, S., Müller, F., Peter, C., Chari, A. and Fischer, U. (2005). Unrip, a factor implicated in cap-independent translation, associates with the cytosolic SMN-complex and influences its intracellular localization. Hum. Mol. Genet 14: 3099-3111. PubMed ID: 16159890
Imlach, W. L., Beck, E. S., Choi, B. J., Lotti, F., Pellizzoni, L. and McCabe, B. D. (2012). SMN is required for sensory-motor circuit function in Drosophila. Cell 151: 427-439. PubMed ID: 23063130
Jablonka, S., Holtmann, B., Meister, G., Bandilla, M., Rossoll, W., Fischer, U. and Sendtner, M. (2002). Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death. Proc. Natl. Acad Sci. 99: 10126-10131. PubMed ID: 12091709
Janice, J., Pande, A., Weiner, J., Lin, C. F. and Makalowski, W. (2012). U12-type spliceosomal introns of Insecta. Int J Biol Sci 8: 344-352. PubMed ID: 22393306
Jodelka, F. M., Ebert, A. D., Duelli, D. M. and Hastings, M. L. (2010). A feedback loop regulates splicing of the spinal muscular atrophy-modifying gene, SMN2. Hum Mol Genet 19: 4906-4917. PubMed ID: 20884664
Kim, E. K., Noh, K. T., Yoon, J. H., Cho, J. H., Yoon, K. W., Dreyfuss, G. and Choi, E. J. (2007). Positive regulation of ASK1-mediated c-Jun NH(2)-terminal kinase signaling pathway by the WD-repeat protein Gemin5. Cell Death Differ. 14: 1518-1528. PubMed ID: 17541429
Kroiss, M., Schultz, J., Wiesner, J., Chari, A., Sickmann, A. and Fischer, U. (2008). Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster. Proc. Natl. Acad. Sci. 105(29): 10045-50. PubMed ID: 18621711
Lee, L., Davies, S. E. and Liu, J. L. (2009). The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway. Dev Biol 332: 142-155. PubMed ID: 19464282
Lee, S., Sayin, A., Grice, S., Burdett, H., Baban, D., et al. (2008). Genome-wide expression analysis of a spinal muscular atrophy model: towards discovery of new drug targets. PLoS ONE 3: e1404. PubMed ID: 18167563
Lefebvre, S., et al. (1995). Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80: 155-165. PubMed ID: 7813012
Liu, Q., and Dreyfuss, G. (1996). A novel nuclear structure containing the survival of motor neurons protein. EMBO J 15: 3555-3565. PubMed ID: 8670859
Liu, Q., Fischer, U., Wang, F., and Dreyfuss, G. (1997). The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell 90: 1013-102. PubMed ID: 9323129
Lotti, F., Imlach, W. L., Saieva, L., Beck, E. S., Hao le, T., Li, D. K., Jiao, W., Mentis, G. Z., Beattie, C. E., McCabe, B. D. and Pellizzoni, L. (2012). An SMN-dependent U12 splicing event essential for motor circuit function. Cell 151: 440-454. PubMed ID: 23063131
Matera, A. G., and Frey, M. R. (1998). Coiled bodies and gems: Janus or gemini? Am. J. Hum. Genet 63: 317-321. PubMed ID: 9683623
McWhorter, M.L., et al. (2003). Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J. Cell Biol. 162: 919-931. PubMed ID: 12952942
Meister, G., et al. (2000). Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins. Hum. Mol. Genet. 9: 1977-1986. PubMed ID: 10942426
Meister, G., et al. (2001). A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs. Nat. Cell Biol. 3: 945-949. PubMed ID: 11715014
Meister, G. and Fischer, U. (2002). Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs. EMBO J. 21: 5853-5863. PubMed ID: 12411503
Miguel-Aliaga, I., Chan, Y. B., Davies, K. E. and van den Heuvel, M. (2000). Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila. FEBS Lett. 486: 99-102. PubMed ID: 11113446
Patel, A. A. and Steitz, J. A. (2003). Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Biol 4: 960-970. PubMed ID: 14685174
Piazzon, N., Rage, F., Schlotter, F., Moine, H., Branlant, C. and Massenet, S. (2008). In vitro and in cellulo evidences for association of the survival of motor neuron complex with the fragile X mental retardation protein. J Biol Chem 283: 5598-5610. PubMed ID: 18093976
Ruggiu, M., McGovern, V. L., Lotti, F., Saieva, L., Li, D. K., Kariya, S., Monani, U. R., Burghes, A. H. and Pellizzoni, L. (2012). A role for SMN exon 7 splicing in the selective vulnerability of motor neurons in spinal muscular atrophy. Mol Cell Biol 32: 126-138. PubMed ID: 22037760
Monani, U. R. (2005). Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron-specific disease. Neuron 48: 885-896. PubMed ID: 16364894
Mouaikel, J., et al. (2003). Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron. EMBO Rep. 4: 616-622. PubMed ID: 12776181
Mouillet, J. F., et al. (2008). DEAD-box protein-103 (DP103, Ddx20) is essential for early embryonic development and modulates ovarian morphology and function. Endocrinology 149: 2168-2175. PubMed ID: 18258677
Nicole, S., et al. (2003). Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle. J. Cell Biol. 161: 571-582. PubMed ID: 12743106
Otter, S., et al. (2007). A comprehensive interaction map of the human SMN complex. J. Biol. Chem. 282: 5825-5833. PubMed ID: 17178713
Praveen, K., Wen, Y. and Matera, A. G. (2012). A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects. Cell Rep 1: 624-631. PubMed ID: 22813737
Praveen, K., Wen, Y., Gray, K. M., Noto, J. J., Patlolla, A. R., Van Duyne, G. D. and Matera, A. G. (2014). SMA-causing missense mutations in Survival motor neuron (Smn) display a wide range of phenotypes when modeled in Drosophila. PLoS Genet 10: e1004489. PubMed ID: 25144193
Rajendra, T. K., Gonsalvez, G. B., Walker, M. P., Shpargel, K. B., Salz, H. K. and Matera, A. G. (2007). A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J. Cell Biol. 176(6): 831-41. PubMed ID: 17353360
Rossoll, W., et al. (2003). Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J. Cell Biol. 163: 801-812. PubMed ID: 14623865
Ruggiu, M., McGovern, V. L., Lotti, F., Saieva, L., Li, D. K., Kariya, S., Monani, U. R., Burghes, A. H. and Pellizzoni, L. (2012). A role for SMN exon 7 splicing in the selective vulnerability of motor neurons in spinal muscular atrophy. Mol Cell Biol 32: 126-138. PubMed ID: 22037760
Schrank, B., Gotz, R., Gunnersen, J. M., Ure, J. M., Toyka, K. V., Smith, A. G. and Sendtner, M. (1997). Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc. Natl. Acad. Sci. 94: 9920-9925. PubMed ID: 9275227
Sen, A., Yokokura, T., Kankel, M. W., Dimlich, D. N., Manent, J., Sanyal, S. and Artavanis-Tsakonas, S. (2011). Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling. J Cell Biol 192: 481-495. PubMed ID: 21300852
Shafey, D., Cote, P. D. and Kothary, R. (2005). Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology. Exp. Cell Res. 311: 49-61. PubMed ID: 16219305
Sharma, A., et al. (2005). A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells. Exp Cell Res. 309: 185-197. PubMed ID: 15975577
Shpargel, K. B. and Matera, A.G. (2005). Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins. Proc. Natl. Acad. Sci. 102: 17372-17377. PubMed ID: 16301532
Shpargel, K. B., Praveen, K., Rajendra, T. K. and Matera, A. G. (2009). Gemin3 is an essential gene required for larval motor function and pupation in Drosophila. Mol. Biol. Cell 20(1): 90-101. PubMed ID: 18923150
Simon, C. M., Janas, A. M., Lotti, F., Tapia, J. C., Pellizzoni, L. and Mentis, G. Z. (2016). A stem cell model of the motor circuit uncouples motor neuron death from hyperexcitability induced by SMN deficiency. Cell Rep 16(5):1416-30. PubMed ID: 27452470
Timmerman, C. and Sanyal, S. (2012). Behavioral and electrophysiological outcomes of tissue-specific Smn knockdown in Drosophila melanogaster. Brain Res 1489: 66-80. PubMed ID: 23103409
Tisdale, S., Lotti, F., Saieva, L., Van Meerbeke, J. P., Crawford, T. O., Sumner, C. J., Mentis, G. Z. and Pellizzoni, L. (2013). SMN is essential for the biogenesis of U7 small nuclear ribonucleoprotein and 3'-end formation of histone mRNAs. Cell Rep 5: 1187-1195. PubMed ID: 24332368; Graphical Abstract
Wan, L., Battle, D. J., Yong, J., Gubitz, A. K., Kolb, S. J., Wang, J. and Dreyfuss, G. (2005). The survival of motor neurons protein determines the capacity for snRNP assembly: biochemical deficiency in spinal muscular atrophy. Mol Cell Biol 25: 5543-5551. PubMed ID: 15964810
Will, C. L. and Luhrmann, R. (2001) Spliceosomal UsnRNP biogenesis, structure and function. Curr. Opin. Cell Biol. 13: 290-301. PubMed ID: 11343899
Winkler, C., Eggert, C., Gradl, D., Meister, G., Giegerich, M., Wedlich, D., Laggerbauer, B. and Fischer, U. (2005). Reduced U snRNP assembly causes motor axon degeneration in an animal model for spinal muscular atrophy. Genes Dev 19: 2320-2330. PubMed ID: 16204184
Winkler, C., et al. (2005). Reduced U snRNP assembly causes motor axon degeneration in an animal model for spinal muscular atrophy. Genes Dev. 19: 2320-2330. PubMed ID: 16204184
Wishart, T. M., Huang, J. P., Murray, L. M., Lamont, D. J., Mutsaers, C. A., Ross, J., Geldsetzer, P., Ansorge, O., Talbot, K., Parson, S. H. and Gillingwater, T. H. (2010). SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy. Hum Mol Genet 19: 4216-4228. PubMed ID: 20705736
Wishart, T. M., et al. (2014). Dysregulation of ubiquitin homeostasis and beta-catenin signaling promote spinal muscular atrophy. J Clin Invest. PubMed ID: 24590288
Yong, J., Kasim, M., Bachorik, J. L., Wan, L. and Dreyfuss, G. (2010). Gemin5 delivers snRNA precursors to the SMN complex for snRNP biogenesis. Mol Cell 38: 551-562. PubMed ID: 20513430
Zhang, H., et al. (2006). Multiprotein complexes of the survival of motor neuron protein SMN with gemins traffic to neuronal processes and growth cones of motor neurons. J. Neurosci. 26: 8622-8632. PubMed ID: 16914688
Zhang, R., So, B. R., Li, P., Yong, J., Glisovic, T., Wan, L. and Dreyfuss, G. (2011). Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly. Cell 146: 384-395. PubMed ID: 21816274
Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M. and Dreyfuss, G. (2008). SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133: 585-600. PubMed ID: 18485868
date revised: 10 October 2014
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