InteractiveFly: GeneBrief

survival motor neuron: Biological Overview | References

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 m^2,2,7G 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).

Modeling spinal muscular atrophy in Drosophila

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 Smn^73Ao 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 Smn^73Ao 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 Smn^73Ao animals (Chan, 2003). It should be noted that other glutamate receptor subunits display altered transcriptional profiles in a Smn^73Ao 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 Smn^73Ao-dependent lethality would be likely to yield genes that also function at the NMJ. Genetic screen using an allele (Smn^73Ao) 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 Smn^73Ao 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).

A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle

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 Smn^E. From a total of ~170 independent excisions, two lines (Smn^E2 and Smn^E33) were isolated that displayed overt motor dysfunction. Smn^E2 and Smn^E33 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 Smn^E33 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 Sm-protein methyl transferase, dart5, is essential for germ-cell specification and maintenance

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 protein methylation is dispensable for snRNP assembly in Drosophila melanogaster

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).

A motor function for the DEAD-Box RNA helicase Gemin3

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 (gemin3^R) or a transheterozygous combination of two transposon inserts which do not complement each other (gemin3^R/gemin3^W) 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).

Gemin3 is an essential gene required for larval motor function and pupation in Drosophila

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 Smn^E33, was created by imprecise excision of a P-element residing in the upstream control region (Rajendra, 2007). Smn^E33 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 Smn^F 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 Smn^E33 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).

Neuromuscular defects in a Drosophila survival motor neuron gene mutant

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)

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 Citation: 17108118

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 Citation: 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 Citation: 15848170

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 Citation: 19023405

Chan, Y. B., et al. (2003). Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum. Mol. Genet. 12: 1367-1376. PubMed Citation: 12783845

Chang, H. C., et al. (2008). Modeling spinal muscular atrophy in Drosophila. PLoS ONE 3: e3209. PubMed Citation: 18791638

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 Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 16129694

Eggert, C., Chari, A., Laggerbauer, B. and Fischer, U. (2006). Spinal muscular atrophy: the RNP connection. Trends Mol. Med. 12: 113-121. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 17895963

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 Citation: 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 Citation: 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 Citation: 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 Citation: 18369183

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 Citation: 16159890

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 Citation: 12091709

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 Citation: 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 Citation: 18621711

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 Citation: 18167563

Lefebvre, S., et al. (1995). Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80: 155-165. PubMed Citation: 7813012

Liu, Q., and Dreyfuss, G. (1996). A novel nuclear structure containing the survival of motor neurons protein. EMBO J 15: 3555-3565. PubMed Citation: 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 Citation: 9323129

Matera, A. G., and Frey, M. R. (1998). Coiled bodies and gems: Janus or gemini? Am. J. Hum. Genet 63: 317-321. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 11113446

Monani, U. R. (2005). Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron-specific disease. Neuron 48: 885-896. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 12743106

Otter, S., et al. (2007). A comprehensive interaction map of the human SMN complex. J. Biol. Chem. 282: 5825-5833. PubMed Citation: 17178713

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 Citation: 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 Citation: 14623865

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 Citation: 9275227

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 Citation: 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 Citation: 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 Citation: 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 Citation: 18923150

Wan, L., et al. (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 Citation: 15964810

Will, C. L. and Luhrmann, R. (2001) Spliceosomal UsnRNP biogenesis, structure and function. Curr. Opin. Cell Biol. 13: 290-301. PubMed Citation: 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 Citation: 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 Citation: 16204184

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 Citation: 16914688

Zhang, Z., et al. (2008). SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133: 585-600. PubMed Citation: 18485868

date revised: 30 January 2010

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