InteractiveFly: GeneBrief

muscleblind: Biological Overview | References


Gene name - muscleblind

Synonyms -

Cytological map position - 54B1-54B5

Function - RNA splice factor

Keywords - alternative splicing, terminal muscle differentiation, eye differentiation, mutants model features of myotonic dystrophy

Symbol - mbl

FlyBase ID: FBgn0265487

Genetic map position - chr2R:13153023-13252891

Classification - C3H1: zinc finger

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Chakraborty, M., Selma-Soriano, E., Magny, E., Couso, J. P., Perez-Alonso, M., Charlet-Berguerand, N., Artero, R. and Llamusi, B. (2015). Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction. Dis Model Mech. PubMed ID: 26515653
Summary:
Up to 80% of myotonic dystrophy type 1 (DM1) patients will develop cardiac abnormalities at some point during the progression of their disease. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. This study describes the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of splice factor Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in human DM1 patients, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function and reduced heart tube diameters and contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, flies were fed with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind and reduced ribonuclear formation in the Drosophila heart but rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.
Cerro-Herreros, E., Fernandez-Costa, J.M., Sabater-Arcis, M., Llamusi, B. and Artero, R. (2016). Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila. Sci Rep 6: 36230. PubMed ID: 27805016
Summary:
Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of extra CTG repeats in the 3' untranslated region (3' UTR). CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Endogenous upregulation of MBNL proteins is, thus, a potential therapeutic approach to DM1. This study identifies two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. It was shown that their sequestration by sponge constructs derepresses endogenous muscleblind not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG trinucleotide repeats throughout the musculature. Enhanced muscleblind expression results in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies have improved muscle function in climbing and flight assays, and have longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.

Chakraborty, M., Sellier, C., Ney, M., Pascal, V., Charlet-Berguerand, N., Artero, R. and Llamusi, B. (2018). Daunorubicin reduces MBNL1 sequestration caused by CUG-repeat expansion and rescues cardiac dysfunctions in a Drosophila model of myotonic dystrophy. Dis Model Mech 11(4). PubMed ID: 29592894
Summary:
Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder caused by expression of mutant myotonin-protein kinase (DMPK) transcripts containing expanded CUG repeats. Pathogenic DMPK RNA sequesters the muscleblind-like (MBNL) proteins, causing alterations in metabolism of various RNAs. Cardiac dysfunction represents the second most common cause of death in DM type 1 (DM1) patients. However, the contribution of MBNL sequestration in DM1 cardiac dysfunction is unclear. This study overexpressed Muscleblind (Mbl), the Drosophila MBNL orthologue, in cardiomyocytes of DM1 model flies and observed a rescue of heart dysfunctions, which are characteristic of these model flies and resemble cardiac defects observed in patients. A drug - daunorubicin hydrochloride - was identified that directly binds to CUG repeats and alleviates Mbl sequestration in Drosophila DM1 cardiomyocytes, resulting in mis-splicing rescue and cardiac function recovery. These results demonstrate the relevance of Mbl sequestration caused by expanded-CUG-repeat RNA in cardiac dysfunctions in DM1, and highlight the potential of strategies aimed at inhibiting this protein-RNA interaction to recover normal cardiac function.

BIOLOGICAL OVERVIEW

Muscleblind-like proteins, Muscleblind (Mbl) in Drosophila and MBNL1-3 in vertebrates, are regulators of alternative splicing. Human MBNL1 is a key factor in the etiology of myotonic dystrophy (DM), a muscle wasting disease caused by the occurrence of toxic RNA molecules containing CUG/CCUG repeats. MBNL1 binds to these RNAs and is sequestered in nuclear foci preventing it from exerting its normal function, which ultimately leads to mis-spliced mRNAs, a major cause of the disease. Muscleblind-proteins bind to RNAs via N-terminal zinc fingers of the Cys(3)-His type. These zinc fingers are arranged in one (invertebrates) or two (vertebrates) tandem zinc finger (TZF) motifs with both fingers targeting GC steps in the RNA molecule. This study shows that mbl genes in Drosophila and in other insects also encode proteins with two TZF motifs, highly similar to vertebrate MBNL proteins. In Drosophila the different protein isoforms have overlapping but possibly divergent functions in vivo, evident by their unequal capacities to rescue the splicing defects observed in mbl mutant embryos. In addition, using whole transcriptome analysis, several new splicing targets were identified for Mbl in Drosophila embryos. Two of these novel targets, kkv (krotzkopf-verkehrt, coding for Chitin Synthase 1) and coracle (coding for the Drosophila homolog of Protein 4.1), are not muscle-specific but expressed mainly in epidermal cells, indicating a function for mbl not only in muscles and the nervous system (Irion, 2012). Drosophila mbl is known to code for several proteins with one N-terminal tandem zinc finger motif; this study found that the genes in Drosophila and in other insects (honey bee, wasp, mosquito) also encode protein isoforms with two TZF motifs, highly similar to the Mbl orthologs in vertebrates. The zinc fingers show a very high degree of conservation between insect Mbl and vertebrate MBNL proteins with almost 80% amino acid similarity. The only significant difference is the spacing between two Cys residues in the second zinc finger of the first TZF motif, with two additional amino acids in insect proteins (Irion, 2012).

Also, the genomic organization of the muscleblind genes is very similar, not only between insects, but also between insects and vertebrates. The intron positions are highly conserved and so is the large size of intron 2. This intron, which splits the coding sequence for the first TZF motif, spans 75 kb in Drosophila and, according to the latest genome assembly, more than 700 kb in honeybees. Nevertheless the spliced product could be easily detected in RNA from pupae (Irion, 2012).

In Drosophila transcripts for a large number of mbl isoforms coding for proteins with one or two TZF motifs are present during all stages of the life cycle. It proved to be difficult to estimate the relative abundance of the different types of transcripts. PCR primers designed to detect both types lead almost exclusively to the amplification of transcripts coding for only one TZF motif. This could be due to much higher abundance of these transcripts, however, it might also reflect a bias in the PCR amplification. Especially because primers designed to detect isoforms with two TZF motifs work very efficiently (Irion, 2012).

There is no indication that vertebrates express MBNL proteins with only one TZF motif. However, for human MBNL1 and MBNL3 it has been shown that truncated versions of the proteins, lacking either one of the two TZF motifs, are still able to bind to RNA and to regulate splicing in a cell culture assay (Grammatikakis, 2011; Irion, 2012 and references therein).

To find additional targets for Mbl in Drosophila the complete transcriptome of stage 16-17 embryos hemizygous for a null-allele was analyzed by Illumina sequencing. Given the fact that a lower accuracy in splice site selection for the α-actinin and ZASP52 transcripts was detected in mutant embryos compared to wild type, the analysis of the RNAseq data focused on the number of total and of unique intron-exon reads as a measure of splicing precision. The known Mbl targets in Drosophila are amongst the highest-ranking transcripts with more than 85 unique intron-exon reads. In the complete list of 81 candidates there is a clear enrichment for the GO annotation terms ‘actin binding’, ‘cytoskeletal protein binding’, ‘protein binding’ and ‘tropomyosin binding’, indicating the prominent function of mbl in muscle development and differentiation. However, many genes with a large number of intron-exon reads represent loci with multiple, often interspersed transcripts, which makes analysis very difficult, e.g. Pde1c, Ect4 or l(3)82Fd. Some genes with many intron-exon reads also harbor transposons, which does account for some of the reads, e.g. Cda5 (Irion, 2012).

Taking these limitations into consideration, the analysis revealed several new potential candidates as targets for Mbl in Drosophila. Ten of these candidates were tested by RT-PCR and splicing defects were found in five of them. The observed defects are either a shift in the ratio of different isoforms (CG33205, cora), the incorrect selection of mutually exclusive exons (wupA, kkv) or general defects in splicing accuracy (Mf) (Irion, 2012).

An important question is whether the different proteins, with one or two TZF motifs and with different C-termini, have different functions in vivo. One possibility could be that all the different Mbl isoforms in Drosophila, which are generated from one gene by extensive alternative splicing, have similar and redundant functions regulating the alternative splicing of the same pre-mRNAs. Another possibility could be that the different proteins have different pre-mRNA targets. As a third alternative some isoforms could be involved in functions altogether different from the regulation of splicing, such as RNA localization. In vertebrates, MBNL proteins are encoded by three separate genes, and for MBNL2 a function in localizing the integrin α3 transcript has been demonstrated (Adereth 2005). Whether any other Mbl protein besides MBNL2 is involved in RNA localization remains to be seen (Irion, 2012).

To address this question the ability was tested of Drosophila Mbl proteins with one or two TZF motifs to rescue the splicing defects occurring in homozygous mutant embryos. It has previously published that Drosophila Mbl regulates alternative splicing of two transcripts in embryos, α-actinin and ZASP52 (Machuca-Tzili, 2006). In homozygous mutant embryos the tissue specific and developmental timing dependent splicing of α-actinin is mis-regulated, e.g., with the premature occurrence of adult isoforms already during embryogenesis. Also in mutant embryos aberrant ZASP52 transcripts can be detected, where a cryptic splice site in exon 15 is used. In both cases there was a clear rescue of the splicing defects by transgene 1, which encodes a protein with only one TZF motif. Transgene 2, which codes for a protein with both TZF motifs, only rescues the α-actinin splicing but not the splicing of ZASP52 (Irion, 2012).

A third published target for Mbl in Drosophila is troponinT (Vicente-Crespo, 2008). It has been shown that in mutant pupae alternative splicing of transcripts from this gene is mis-regulated. Also in embryos there is a shift in the ratio of two different isoforms of troponinT transcripts in mbl mutants. This defect is not rescued by either of the two transgenes tested (Irion, 2012).

Having identified additional transcripts mis-spliced in mbl mutant embryos, the two different transgenes were tested for their ability to rescue these defects. There is generally good rescue with transgene 1 whereas transgene 2 only rescues the splicing of cora and kkv to some extent (Irion, 2012).

The observed differences between the two transgenes indicate that there might be functional differences between Mbl proteins with one and two TZF motifs. Both are clearly regulators of alternative splicing, as they efficiently rescue the splicing of α-Actinin, which also demonstrates that expression occurs from both transgenes and that the proteins function in the nucleus. The different rescue capacities for the other mbl targets could indicate different specificities of the protein isoforms, where the protein with two TZF motifs might have other, not yet identified, targets. An alternative explanation, which cannot be ruled out at this stage, is that the proteins are present in the cells in very different amounts or that they localize mostly to different sub-cellular compartments. Because both transgenes are inserted at the same genomic location on the X-chromosome and the same GAL4 driver-line was used it seems unlikely that significantly different RNA levels should be transcribed. Therefore one would have to assume that the translation efficiencies of the RNAs are different, or that the extra 121 amino acid residues affect the stability or localization of the protein encoded by transgene 2. This hypothesis will only be testable after generation of an antibody, which allows the detection of Mbl proteins at endogenous levels on western blots (Irion, 2012).

Apart from CUG repeats, the five nucleotide sequence, 5'-AGUCU-3', has been identified as a consensus binding motif for Drosophila Mbl by in vitro-studies using a protein with the first TZF motif (Goers, 2008). This sequence motif occurs, however, only rarely in intronic sequences flanking those exons that are mis-spliced in mbl mutants. It has also been suggested that Mbl recognizes complex RNA secondary structures, which would not be easy to predict (Irion, 2012).

In contrast, it was found that expression of the human gene, MBNL1, can rescue embryonic lethality in Drosophila, suggesting that both proteins recognize the same target RNAs (Monferrer, 2006). In vitro selection has lead to the definition of a binding motif for the human protein, which is a GC motif embedded in pyrimidines, 5'-YGCY-3', with a preference for at least one pyrimidine-base (Y) being U (Goers, 2008). In efficient high-affinity binding sites this motif often occurs in several copies in the RNA. In a simple model for the regulation of alternative splicing, exon exclusion is promoted when MBNL binds upstream of the exon, whereas binding downstream of the exon enhances its inclusion (Irion, 2012).

Given the high degree of similarity between the zinc finger motifs in Drosophila Mbl and human MBNL1 it seems likely that the Drosophila protein recognizes the same binding sites in pre-mRNAs. Analysis of the complete Drosophila genome showed that the binding motif occurs on average 1.72 times in intronic sequences close to exons (<200 bp) (Vipin T. Sreedharan and Gunnar Rätsch, personal communication to Irion, 2012). In the intronic sequences close to the exons 5 and 6 in Actn there are indeed clusters of YGCY motifs. In this case there is also good correlation with the model that Mbl binding downstream enhances inclusion (exon 5a) whereas binding upstream of the exon predominantly leads to exclusion (exon6). For the other exons where Mbl regulates splicing, mostly identified in this study, the correlation is less clear. For example, in the case of wupA there is no clustering of YGCY motifs detectable, although the presence of Mbl protein clearly leads to the inclusion of exon 6a and the exclusion of exons 6b-6d. In several other cases it is not simple the inclusion or exclusion of an exon that is regulated by Mbl, but the selection between alternative splice sites for one exon (Irion, 2012).

Drosophila is an important model organism to study the function of Mbl proteins in vivo during development and their role in the pathogenesis of myotonic dystrophy. This study has shown that the fly gene codes for proteins that are more similar to the human proteins than previously recognized. The different protein isoforms seem to have distinct functions in the regulation of alternative splicing. Further analysis will be needed to understand the in vivo contributions of the different protein isoforms towards the regulation of alternative splicing and possibly in other processes (Irion, 2012).

Two Enhancers Control Transcription of Drosophila muscleblind in the Embryonic Somatic Musculature and in the Central Nervous System

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In this study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene (Bargiela, 2014).

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis

Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene. This study used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematodes to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors leads to an externally rough eye morphology. This phenotype has been used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog arrest, exon junction complex components tsunagi and always early, and pro-apoptotic genes Traf1 and reaper. This study further investigated Muscleblind implication in apoptosis and splicing regulation. Missplicing of troponin T was found in muscleblind mutant pupae, and Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing was confirmed in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression. Taken together these genetic approaches identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell (Vicente-Crespo, 2008).

Using Drosophila as a model organism, this study reports the first screen specifically addressed to identify gene functions related to the biomedically important protein Muscleblind. In support of the relevance of the results, the strong functional conservation between fly and vertebrate Muscleblind proteins is shown. Furthermore, data is presented supporting that Muscleblind can induce apoptosis in vivo in imaginal disc tissue, and a conserved motif in the MblC protein isoform was identified that conferred pro-apoptotic activity in Drosophila cell culture when mutated. Noteworthy, this is the first conserved motif (besides CCCH zinc fingers) that is associated with a particular function in Muscleblind proteins (Vicente-Crespo, 2008).

Whereas most vertebrates include three muscleblind paralogues in their genomes, a single muscleblind gene carries out all muscleblind-related functions in Drosophila. These functions are probably accomplished through alternative splicing, which generates four Muscleblind protein isoforms with different carboxy-terminal regions. An evolutionary analysis was performed with isoform-specific protein sequences in order to assess conservation of alternative splicing within protostomes. MblC-like isoforms have been detected even in the nematodes C. elegans and Ascaris suum but not MblA, B or D, that were only consistently found within Drosophilidae. Interestingly, also vertebrate Mbnl1 genes included MblC-like sequences. This finding, together with previous studies that shown that mblC is the isoform with the strongest activity in a muscleblind mutant rescue experiment and α-actinin minigene splicing assay (Vicente, 2007) point to mblC as the isoform performing most of muscleblind functions in the fly. Despite this, Muscleblind isoforms are partially redundant. Both mblA and B partially rescue the embryonic lethality of muscleblind mutant embryos (Vicente, 2007) and were able to similarly promote foetal exon exclusion in murine TnnT3 minigene splicing assays. MblD showed no activity in splicing assays or in vivo overexpression experiments. However, we show a marginal increase in cell viability in cell death assays. Using isoform-specific RNAi constructs we plan to re-evaluate the function of Muscleblind isoforms both in vivo and in cell culture (Vicente-Crespo, 2008).

Although the regulation of alternative splicing by Muscleblind proteins is an established fact, the cellular processes in which the protein participates are largely unknown. Genetic screens provide a way to approach those processes as they interrogate a biological system as a whole. Overexpression of MblC in the Drosophila eye originated an externally rough eye phenotype that is temperature sensitive, thus indicating sensitization to the muscleblind dose. A deficiency screen was performed, and several candidate mutations were tested for dominant modification of the phenotype. Nineteen were identifed genes of which more that half can be broadly classified as involved in apoptosis regulation (rpr, th and Traf1), RNA metabolism (Aly, tsu, aret and nonA) or transcription regulation (jumu, amos, Dp, CG15435 and CG15433), whereas the rest do not easily fall into defined classes. muscleblind has been shown to regulate α-actinin and troponinT alternative splicing both in vivo and in cell culture (Vicente, 2007; Machuca-Tzili, 2006). The genetic interaction with the Drosophila homolog of human splicing factor CUG-BP1 (aret) and nonA supports a functional relationship in flies. The antagonism between MBNL1 and CUG-BP1 has actually been shown in humans (Ladd, 2005), whereas RNA-binding protein NonA might be relevant to Muscleblind sequestration by CUG repeat RNA in flies (Houseley, 2005; Vicente-Crespo, 2008 and references therein).

Reduction of dose of exon junction complex (EJC) components tsunagi and Aly also modify MblC overexpression phenotype. EJC provides a binding platform for factors involved in mRNA splicing, export and non-sense mediated decay (NMD). This suggests a previously unforeseen relationship between Muscleblind and EJC, perhaps helping to couple splicing to mRNA export. Consistently, Aly mutations enhanced a CUG repeat RNA phenotype in the Drosophila eye. A similar coupling between transcription and splicing might explain the identification of a number of transcription factors in the screen. Of these, the effect of jumu alleles in the eye and wing MblC overexpression phenotypes were studied in some detail. Loss of function jumu mutations suppress both wing defects and rough eye, whereas they have no effect on unrelated overexpression phenotypes thus suggesting that the interaction is specific (Vicente-Crespo, 2008).

Mutations in the Drosophila homolog of vertebrate Inhibitor of Apoptosis (Diap1 or thread) dominantly enhanced the rough eye phenotype. Consistently with the specificity of the interaction, a second Drosophila paralog, Diap2, did not interact. Also, a deficiency that removes the Drosophila proapoptotic genes hid, reaper and grim (which inhibit thread) was a dominant suppressor while reaper overexpression in eye disc enhanced the phenotype. Interestingly the human homolog of Drosophila Hsp70Ab, Hsp70, has been related to apoptosis as it directly interacts with Apaf-1 and Apoptosis Inducing Factor (AIF) resulting in the inhibition of caspase-dependent and caspase-independent apoptosis. All these genetic data are consistent with MblC overexpressing eye discs being sensitized to enter apoptosis, although no increase in caspase-3 activation was detected in third instar eye imaginal disc overexpressing MblC (Vicente-Crespo, 2008).

Human MBNL1 and CUB-BP1 cooperate to regulate the splicing of cardiac TroponinT (cTNT, Ho, 2007). The current study detected splicing defects in Drosophila troponinT mRNA in muscleblind mutant pupae. Interestingly, an abnormal exclusion of exon 3 was detected in muscleblind mutant pupae, encoding a glutamic acid-rich domain homologous to the foetal exon of cTNT regulated by human MBNL1 (Benoist, 1998). Drosophila exon 3 is only absent in the troponinT isoform expressed in TDT and IFM muscles and probably confers specific functional properties much like the foetal exon does in humans (Chaudhuri, 2005). This identifies troponinT as a new target of Muscleblind activity in flies (Vicente-Crespo, 2008).

CUG-BP1 protein antagonizes MBNL1 exon choice activity in IR and cTNT pre-mRNAs. Moreover, a genetic interaction has been detected between MblC overexpression and aret loss of function mutations. In order to further characterize the functional interaction between Muscleblind and Bruno proteins, their ability to regulate murine TnnT3 was examined in human cell culture. MblA, B and C showed strong activity on TnnT3 mRNA but no significant activity was detected for any Bruno protein. This shows a strong functional conservation between fly and vertebrate Muscleblind proteins as Drosophila isoforms can act over a murine target in a human environment. In contrast, Bruno proteins might not conserve the regulatory activity over troponinT mRNA described for their vertebrate homologues or at least they were not functional in the cellular environment used in this assay. Because GFP-tagged Bruno proteins were only weakly expressed in HEK cells under the experimental conditions used, the level of expression might be insufficient to overcome endogenous Muscleblind activity in cell culture. Furthermore, Bruno proteins might antagonize Muscleblind on a different subset of RNA targets. Although bruno1 has been shown to regulate splicing of some transcripts in S2 cell culture and Bruno3 binds the same EDEN sequence than human CUG-BP, no in vivo experiments have addressed the functional conservation between fly and vertebrate Brunos. Bruno1 is expressed in the germ line where it acts as translational repressor of oskar and gurken mRNAs (Vicente-Crespo, 2008).

Wing imaginal discs stained with anti-caspase-3 and with TUNEL showed that activation of apoptosis was not general in cells expressing MblC but restricted to defined regions within the disc, in particular the wing blade. The spatial constraints that were observed within the imaginal disc might explain the small effect detected when expressing Muscleblind proteins in S2 cells. MblC might require the presence of other factors to be able to unleash programmed cell death. Alternatively, the level of overexpression may be critical and transfected Muscleblind proteins may not reach a critical threshold in Drosophila S2 cells. MblC activation of apoptosis could reveal a direct regulation of apoptotic genes at RNA level or be an indirect effect. Several apoptotic genes produce pro-apoptotic or anti-apoptotic isoforms depending on the regulation of their alternative splicing. MblC could be similarly regulating protein isoforms originating from one or a number of key apoptotic genes at the level of pre-mRNA splicing. Alternatively, MblC could be regulating isoform ratio of a molecule indirectly related to programmed cell death, for example a cell adhesion molecule causing apoptosis by inefficient cell attachment to the substrate. Furthermore, human MBNL proteins are implicated not only in splicing but also in RNA localization, a process that if conserved in flies can potentially impinge in apoptosis regulation (Vicente-Crespo, 2008).

The analysis of MblC-specific sequence revealed a region conserved in Muscleblind proteins from nematodes to humans. Post-translational prediction programs found a motif (FKRP) weakly resembling a sumoylation target site. However, results in S2 cells suggest that sumoylation, if actually taking place, modifies only a small fraction of MblC proteins. FKRP may alternatively participate in an interaction with a Muscleblind partner potentially regulating activity or location in cell compartments, assist in protein dimerization, or others functions. The FKRP site was mutated and a number of functional assays were performed using the mutant MblC. Whereas MblCK202I excluded foetal exon in TnnT3 minigene splicing assays and bound CUG repeat RNA like its wild type counterpart, the mutant protein showed a different preferential distribution in human cells and significantly increased cell death activation upon overexpression. The mechanism by which the FKRP site influences subcellular distribution and cell death-inducing activities is currently unknown, but nevertheless constitutes the first motif, other than zinc fingers, that is associated with a function within Muscleblind proteins (Vicente-Crespo, 2008).

Genetic analysis of chaste, a new mutation of Drosophila melanogaster characterized by extremely low female sexual receptivity

By screening about 2,000 P-element-insertion lines of Drosophila melanogaster, a new behavioral mutant line, chaste (chst), the females of which display extraordinarily strong rejection behavior against courting males. The chst mutation mapped to the muscleblind (mbl) locus at 54B on the right arm of chromosome 2. The reduced sexual receptivity in chst mutant females is reversed to the wild-type level by introducing a transgene, which expresses either the mblB+ or mblC+ isoform, demonstrating that chst is an allele of mbl. Among the P-elements inserted upstream of the mbl gene, those inserted in the same orientation as that of mbl express the chst phenotype, whereas a P-element inserted in the opposite orientation does not. This finding implies that the former P-elements induce the mutant phenotype by a mechanism that is sensitive to the direction of transcription (e.g., transcriptional interference). The mbl alleles, with deletions near the transcription start site and/or in part of the exons, complement the chst mutation in the sexual receptivity phenotype, but not in the lethality phenotype of mbl mutations. Such interallelic complementation of the sexual receptivity phenotype in the mbl locus disappears in the presence of a mutant copy of zeste (z), a gene encoding a protein that mediates transvection. It is suggested that the mbl gene function is required for the normal development of neural substrates that regulate female sexual receptivity (Juni, 2009).

A conserved motif controls nuclear localization of Drosophila Muscleblind

Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and it has been determined that these have different subcellular localizations in the COS-M6 cell line. This study describes the conservation of the sequence motif KRAEK in isoforms C and E and proposes a specific function for this motif. Different Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear or cytoplasmic compartment (MblC and MblD) in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization, whereas fusion of a single KRAEK motif to the heterologous protein beta-galactosidase is sufficient to target the reporter protein to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types. Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute a new class of nuclear localization signal (Fernandez-Costa, 2010).

Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model

Myotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, a genetic screen was carried using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology (Llamusi, 2013).

Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Recent studies have also reported miRNA dysregulation in DM1 patients. In this work, a Drosophila model was used to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were differentially expressed in CTG-expressing flies. Of these, 19 were down-regulated, whereas 1 was up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs were up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 included a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) was also suggested for miR-1 and miR-7, as these miRNAs were down-regulated in flies where Mbl had been silenced. Finally, the physiological relevance of miRNA dysregulation was demonstrated for miR-10, since over-expression of this miRNA in Drosophila extended the lifespan of CTG-expressing flies. Taken together, these results contribute to understanding of the origin and the role of miRNA alterations in DM1 (Fernandez-Costa, 2013).

Ubiquitous expression of CUG or CAG trinucleotide repeat RNA causes common morphological defects in a Drosophila model of RNA-mediated pathology

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington's disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their 'molecular hallmarks' that can be used to determine the contribution of each pathway in human pathology. This study characterised a tergite disruption phenotype in adult flies caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait a new genetic system was identified in which to examine 'hairpin' repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model. Mbl sequestration did not appear to be a major contributor to the common pathway that leads to similar CUG or CAG-mediated tergite disruption. CAG transcripts in this system that did not form specific foci, gave a strong tergite phenotype, and thus are able to undergo as yet undefined interactions necessary to induce pathology. These results, together with observations of sequence specific modification with Mbl and Dcr-1, are consistent with the possibily that 'hairpin' repeat RNA may undergo multiple interactions that contribute to pathology (Lawler, 2012).

Genetic and chemical modifiers of a CUG toxicity model in Drosophila

Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL) proteins contributing to myotonic dystrophy 1 (DM1). To understand this toxic RNA gain-of-function mechanism a Drosophila model was developed expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen), muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine), and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments (Garcia-Lopez, 2008).

Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins

The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally deplete Mbnl proteins. This study identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis (Wang, 2012).

RNA binding specificity of Drosophila muscleblind

Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. This study reports the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-stranded CUG repeats demonstrated that Mbl requires pyrimidine-pyrimidine mismatches for binding and that the identity and location of the C-G and G-C base pairs within the repeats are essential for Mbl binding. Systematic evolution of ligands by exponential enrichment (SELEX) was used to identify RNA sequences that bind Mbl with much higher affinity than CUG repeats. The RNA sequences identified by SELEX are structured and contain a five-nucleotide consensus sequence of 5'-AGUCU-3'. RNase footprinting of one of the SELEX RNA sequences with Mbl showed that Mbl binds both double-stranded and single-stranded regions of the RNA. Three guanosines show the strongest footprint in the presence of Mbl; mutation of any of these three guanosines eliminates Mbl binding. It was also found that Mbl specifically bound a human MBNL1 RNA target, demonstrating the conservation of the muscleblind proteins in recognizing RNA targets. These results reveal that Mbl recognizes complex RNA secondary structures (Goers, 2008).

Expression pattern of muscleblind-like proteins differs in differentiating myoblasts

Muscleblind-like (MBNL) proteins are believed to be regulators of myogenesis and are implicated in myotonic dystrophy. While Drosophila melanogaster muscleblind is required for terminal muscle differentiation, mammalian MBNL3 functions as an inhibitor of myogenesis. This study analyzed the expression pattern of MBNL3 in different adult mouse tissues and tissue culture cells. MBNL3 transcript is enriched in the lung, spleen, and testis and not in heart and skeletal muscle. By Western blotting, it was found that MBNL3 was expressed in C2C12 myoblasts and ts13 myofibroblasts, but was detected at significantly lower levels in fibroblasts. MBNL3 protein levels decreased when cells were shifted to muscle differentiation conditions, but the closely related MBNL1 protein was unaffected. These results suggest that myoblasts and fibroblasts respond to differentiation conditions by activating signaling pathways that repress MBNL3 but not MBNL1 expression (Lee, 2007).

Muscleblind isoforms are functionally distinct and regulate alpha-actinin splicing

Drosophila Muscleblind (Mbl) proteins control terminal muscle and neural differentiation, but their molecular function has not been experimentally addressed. Such an analysis is relevant as the human Muscleblind-like homologs (MBNL1-3) are implicated in the pathogenesis of the inherited muscular developmental and degenerative disease myotonic dystrophy. The Drosophila muscleblind gene expresses four protein coding splice forms (mblA to mblD) that are differentially expressed during the Drosophila life cycle, and which vary markedly in their ability to rescue the embryonic lethal phenotype of muscleblind mutant flies. Analysis of muscleblind mutant embryos reveals misregulated alternative splicing of the transcripts encoding Z-band component alpha-Actinin, which can be replicated in human cells expressing a Drosophila alpha-actinin minigene and epitope-tagged Muscleblind isoforms. MblC appreciably altered alpha-actinin splicing in this assay, whereas other isoforms had only a marginal or no effect, demonstrating functional specialization among Muscleblind proteins. To further analyze the molecular basis of these differences, the subcellular localization of Muscleblind isoforms was studied. Consistent with the splicing assay results, MblB and MblC were enriched in the nucleus while MblA was predominantly cytoplasmic. In myotonic dystrophy, transcripts bearing expanded non-coding CUG or CCUG repeats interfere with the function of human MBNL proteins. Co-expression of CUG repeat RNA with the alpha-actinin minigene altered splicing compared with that seen in muscleblind mutant embryos, indicating that CUG repeat expansion RNA also interferes with Drosophila muscleblind function. Moreover MblA, B, and C co-localize with CUG repeat RNA in nuclear foci in cell culture. These observations indicate that Muscleblind isoforms perform different functions in vivo, that MblC controls muscleblind-dependent alternative splicing events, and establish the functional conservation between Muscleblind and MBNL proteins both over a physiological target (alpha-actinin) and a pathogenic one (CUG repeats) (Vicente, 2007).

Muscleblind proteins regulate alternative splicing

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. This study demonstrated that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets (Ho, 2004).

Noncanonical RNAs from transcripts of the Drosophila muscleblind gene

It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. This study describes new cDNAs from the Drosophila melanogaster muscleblind (mbl) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2') or having exons with a different order compared to the corresponding genomic DNA (mblE2E3'E2'; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomplete exon 2 tandem repetition was confirmed by sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products, rapid amplification of cDNA ends, and detection of a band consistent with cDNA sizes in total RNA northern blots. RT-PCRs with exon-specific primers downstream of exon 2 were unable to amplify products other than those expected from canonical mbl isoforms, thus indicating that no other exons were efficiently spliced downstream of exon 2. Moreover, mblE2E2' transcripts seem to be poorly polyadenylated, if at all, and behave aberrantly in a polyacrylamide gel electrophoresis (PAGE) mobility assay. Taken together, lack of polyadenylation, lack of downstream splicing events, small size of mblE2E2', and PAGE behavior all suggest that these noncanonical transcripts may be circular RNAs. The functional implications for these noncanonical transcripts are unclear. A developmental expression profile of mblE2E2' revealed an almost constant expression except during early embryogenesis and early adulthood. The protein putatively encoded is unlikely to be functional because an in-frame stop codon occurs almost immediately after the splice site. Such noncanonical transcripts have previously been observed in vertebrates, and these data provide the first experimental evidence for similar phenomena in invertebrates (Houseley, 2006).

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. This study, using extensive mutagenesis of expression plasmids and RNAi screening, revealed that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (approximately 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, it was found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes (Kramer, 2015).

It was long assumed that eukaryotic pre-mRNAs are always canonically spliced to generate a linear mRNA that is subsequently translated to produce a protein. However, it is now becoming increasingly clear that many genes can be noncanonically spliced to produce circular RNAs with covalently linked ends. These transcripts are almost exclusively derived from exons, accumulate in the cytoplasm, and are thought to be products of alternative splicing events known as 'backsplicing.' In contrast to canonical splicing, which joins the exons in a linear order (joining exon 1 to exon 2 to exon 3, etc.), backsplicing joins a splice donor to an upstream splice acceptor (e.g., joining the 3' end of exon 2 to the 5' end of exon 2). A handful of RNAs generated in this manner were identified in the 1990s, and recent deep sequencing studies have expanded this observation to thousands of circular RNAs expressed across eukaryotes, including humans, Caenorhabditis elegans, Drosophila (Salzman. 2013; Ashwal-Fluss, 2014; Westholm, 2014), Schizosaccharomyces pombe, and plants. Perhaps surprisingly, for some genes, the abundance of the circular RNA exceeds that of the associated linear mRNA by a factor of 10, suggesting that the major function of some protein-coding genes may be to generate circular RNAs (Kramer, 2015).

Most exons in eukaryotic genomes have splicing signals at both ends and theoretically can circularize. However, only certain exons are observed in circular RNAs, and these backsplicing events often occur in a tissue-specific manner. This suggests that circular RNA biogenesis is tightly regulated. As splicing generally occurs cotranscriptionally, most introns, along with their upstream splice acceptors (which are needed for backsplicing), are rapidly removed. Therefore, for circular RNAs to be produced, canonical splicing likely must occur more slowly around these exons, and/or exon skipping events may be coupled to circular RNA biogenesis. In the latter, the circular RNA is derived from an exon-containing lariat, allowing a pre-mRNA to yield both a linear mRNA and a circular RNA comprised of the skipped exons (Kramer, 2015).

There is little known about the splicing factors that regulate these events. In some cases, the Muscleblind (Mbl) and Quaking proteins appear to facilitate backsplicing by bridging between two introns and causing the splice sites from the intervening exons to be brought into close proximity (Ashwal-Fluss, 2014; Conn, 2015). For example, circular RNA production from the Drosophila mbl gene is triggered when the Mbl splicing factor binds to its own introns (Ashwal-Fluss, 2014). However, in humans, mice, and C. elegans, the predominant determinants of whether a pre-mRNA is subjected to backsplicing are intronic repetitive elements, such as sequences derived from transposons. Almost 90% of human circular RNAs have complementary Alu elements in their flanking introns, and, analogous to the protein-bridging mechanism, base-pairing between complementary sequences allows the intervening splice sites to be brought close together. Interestingly, repeats <40 nucleotides (nt) can drive circular RNA production in human cells, but it is clear that more than simple thermodynamics regulates circularization. For example, base-pairing interactions can be disrupted by ADAR (adenosine deaminase acting on RNA), which converts adenosines in double-stranded regions to inosines. In addition, most mammalian pre-mRNAs contain multiple intronic repeats, allowing distinct circular (or linear) RNAs to be produced depending on which repeats base-pair to one another. Therefore, other factors likely help dictate splicing outcomes by regulating these exon circularization events (Kramer, 2015).

Despite key regulatory roles for intronic repeats in multiple eukaryotes, it has been suggested that circular RNA biogenesis in Drosophila melanogaster is not driven by base-pairing interactions (Westholm, 2014). Instead, a positive correlation between the length of the flanking introns and circular RNA abundance was identified in Drosophila (Westholm, 2014). However, the effect of modulating intron lengths on backsplicing has not yet been directly addressed. It is also completely unknown how Drosophila circular RNAs besides Mbl, of which there are >2500 annotated circular RNAs derived from other genomic loci, are generated or post-transcriptionally regulated. Therefore, it is still unclear whether circular RNA biogenesis strategies are conserved across eukaryotes or whether species such as Drosophila use unique mechanisms to determine which exons should be backspliced (Kramer, 2015).

Once produced, circular RNAs are stable transcripts that are naturally resistant to degradation by exonucleases. Two circular RNAs (ciRS7/CDR1as and Sry) modulate the activity of specific microRNAs (Hansen, 2013; Memczak, 2013), but most other RNA circles (in species other than Drosophila) contain few microRNA-binding sites and likely function differently. For example, it has been proposed that many circular RNAs may regulate neuronal functions, and artificial circular RNAs containing an IRES (internal ribosome entry site) can be translated. However, the lack of efficient methods for modulating circular RNA levels or ectopically expressing circular RNAs has limited the ability to define functions for these transcripts (Kramer, 2015).

This study focused on the Drosophila laccase2 gene, as it produces an abundant circular RNA in vitro and in vivo. Evidence is provided that intronic repeats collaborate with trans-acting splicing factors to regulate circularization in flies. Mechanistically, it was found that miniature introns (<150 nt) containing the splice sites and inverted repeats were sufficient to support Laccase2 circular RNA production. The intronic repeats must base-pair to one another for circularization to occur, as has been observed in other eukaryotes. Furthermore, it was found that the strength of these base-pairing interactions dictates whether backsplicing occurs co- or post-transcriptionally: Long flanking repeats appear to allow cotranscriptional processing. Screening a panel of genes, this study found that multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine–arginine) family proteins regulate Laccase2 circular RNA levels in a combinatorial manner. Comparisons with the mbl locus suggest that the circularization mechanisms are distinct, as the Laccase2 circular RNA was not regulated by the Mbl or Laccase2 gene products. Additional circular RNAs were identified that are regulated by unique combinations of hnRNP and SR proteins, suggesting that combinatorial control may be a common regulatory strategy that modulates circular RNA levels. This led to a test of whether this biogenesis mechanism is active in human cells, and it was found that the laccase2 introns can indeed robustly generate circular RNAs. It is thus now possible to efficiently generate "designer" circular RNAs in cells with minimal linear RNA production. In total, the results reveal new insights into how trans-acting factors and intronic repeats collaborate to regulate circular RNA biogenesis across eukaryotes as well as provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

This study demonstrates that intronic repeats and trans-acting hnRNPs and SR proteins combinatorially regulate circularization of the Drosophila laccase2 gene. Base-pairing between transposable elements in the flanking introns facilitates circularization, and the strength of these interactions likely dictates whether backsplicing occurs co- or post-transcriptionally. This mechanism is distinct from the one that regulates Drosophila Mbl circular RNA production (Ashwal-Fluss, 2014) but is similar to that used to generate many circles in humans, mice, and C. elegans. This suggests that base-pairing between intronic repeats may be a major mechanism promoting exon circularization across eukaryotes. Moreover, this study found that the laccase2 exon is dispensable, allowing the laccase2 introns to be used to efficiently generate 'designer' circular RNAs from plasmids in diverse organisms. Altogether, the results suggest that circular RNA biogenesis strategies are conserved across eukaryotes and provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

The current results on the laccase2 locus indicate that base-pairing between complementary intronic sequences efficiently promotes RNA circularization in flies. As the DNAREP1_DM repeats closely flank exon 2 of the laccase2 gene, a model is proposed in which the repeats base-pair to one another, bringing the intervening splice sites into close proximity and facilitating catalysis. The Laccase2 circular RNA then accumulates as one of the most abundant circular RNAs in Drosophila (fifth most abundant across >100 Drosophila RNA sequencing libraries). At the endogenous laccase2 gene locus, the long introns that flank this exon likely slow the overall speed of cotranscriptional splicing, thereby allowing the backsplicing reaction to effectively compete with canonical splicing. Indeed, it was found that the strength of the base-pairing interactions between the flanking introns dictates how quickly backsplicing can occur. When very stable interactions are present, it is possible that exon definition is improved, allowing the rapid and cotranscriptional generation of a circular RNA. Nevertheless, further studies are still required to clarify the exact role that long flanking introns may play in regulating circularization (Kramer, 2015).

Upon examining the introns that flank other abundant Drosophila circular RNAs, this study identified other examples in which complementary regions >60 nt in length flank circularizing exons, including CaMKI, CG11155, CG2052, Parp, and PlexA (which are among the top 25 most abundant Drosophila circular RNAs). Interestingly, the Semaphorin-2b (CG33960) circular RNA (39th most abundant circular RNA) is flanked by introns containing short (CA)n simple repeats that are complementary to each other over a <30-nt region. Upon cloning a 980-nt region of the Semaphorin-2b pre-mRNA downstream from the pMT, circular RNA production from the plasmid was observed in DL1 cells. Removal of either of the (CA)n simple repeats, however, strongly reduced circularization. This suggests that diverse inverted repeat sequences, including short simple repeats, may play a general role in facilitating circularization in Drosophila (Kramer, 2015).

Complementary repeats, however, are not observed at all Drosophila loci that generate circular RNAs. Furthermore, many exons that do not circularize are flanked by complementary repeats, so there must be other mechanisms that regulate circularization. This has been most notably demonstrated at the Drosophila mbl locus, which requires the Mbl splicing factor for its circularization. When Mbl protein is in excess, an intricate feedback mechanism is induced: The Mbl protein decreases the production of its own mRNA by binding its pre-mRNA. This blocks canonical splicing and promotes the biogenesis of the Mbl circular RNA, which further functions as a sponge that binds and sequesters the excess Mbl protein. However, this Mbl-driven mechanism appears to be specific for the mbl locus, as this study found that knockdown of the Mbl linear mRNA had no effect on Laccase2, PlexA, or a panel of other circular RNAs. Knockdown of the Laccase2 linear mRNA likewise did not affect Laccase2 circular RNA levels, indicating that the laccase2 locus is not subjected to a similar direct cis-acting feedback mechanism. Instead, it was found that other splicing factors, including hnRNPs and SR proteins, regulate Laccase2 RNA levels (Kramer, 2015).

At the laccase2 locus, it is proposed that hnRNPs (e.g., Hrb27C and Hrb87F) and SR proteins (e.g., SF2 [SRSF1], SRp54 [SRSF11], and B52 [SRSF6]) add an additional layer of control on top of the DNAREP1_DM intronic repeats. Base-pairing between the intronic repeats promotes circularization, but protein binding likely helps ensure that the appropriate ratio of linear to circular Laccase2 RNA is produced. Depletion of any one of these splicing factors alters Laccase2 circle levels, and additive effects were observed when multiple factors were depleted. This suggests combinatorial control, with each protein playing a nonredundant role. Furthermore, Laccase2 circular RNA production does not appear to be linked to exon skipping, and thus these proteins may specifically modulate spliceosome assembly, the speed of splicing, and/or the stability of the mature circular RNA. Notably, it does not seem that Hrb27F, SF2, SRp54, or B52 affects Laccase2 circular RNA stability, as depletion of these factors did not cause the expression of a plasmid-derived Laccase2 circular RNA to increase. It is thus instead proposed that these hnRNPs and SR proteins regulate Laccase2 circular RNA biogenesis (e.g., by binding to the flanking introns or exons), but further studies are required to understand exactly how the intronic repeats and trans-acting factors collaboratively dictate the splicing outcome. Nevertheless, the same SR proteins that regulate the laccase2 locus also regulate the PlexA circular RNA but not the Mbl circular RNA. Since the laccase2 and PlexA exons are both flanked by inverted repeats, it is hypothesized that intronic repeats may generally provide the opportunity for circularization to occur. This is then further regulated by trans-acting factors that combinatorially fine-tune the amount of each circular RNA that the cell ultimately produces (Kramer, 2015).

Catalogs of circular RNAs expressed in various species and cell types have been reported, but the functions for nearly all of these transcripts, including Laccase2, are currently unknown. This is due in part to the current lack of methods for efficiently generating circular RNAs in cells. For example, the circular RNA expression plasmids that have been described all generally produce circular transcripts at a low efficiency (often 20% or less). These plasmids instead generate abundant amounts of linear RNA, which limits their utility for defining circular RNA functions. Using the Drosophila laccase2 and human ZKSCAN1 introns, this study largely overcame this hurdle and generated circular RNAs (ranging in size from 300 to 1500 nt) at a high efficiency in human and fly cells. These transcripts accumulate in the cytoplasm, are resistant to RNase R treatment, and are likely translated when an IRES is present. Furthermore, easy-to-use restriction sites are present in the plasmids, allowing any desired sequence to be queried. Beyond allowing ectopic expression of circular RNAs, these plasmids can be designed to sponge microRNAs or proteins as well as identify novel IRES sequences (Kramer, 2015).

In summary, the current findings provide key insights into how trans-acting factors and intronic repeats regulate circular RNA biogenesis as well as provide new tools for exploring the functions of circular RNAs across eukaryotes. From humans to flies, repetitive elements in introns can act to facilitate backsplicing, but it is still largely unclear why circular RNAs accumulate only in certain tissues. It is hypothesized that base-pairing between repeats is only one part of the "splicing code", and it is ultimately a combination of cis-acting elements and trans-acting splicing factors, including hnRNPs and SR proteins, that dictates whether canonical splicing or backsplicing occurs. Nevertheless, this study has defined a minimal set of elements that is sufficient for promoting efficient exon circularization, which should facilitate the prediction of circular RNAs as well as enable the functions of many circular RNAs to be revealed. Considering that a surprisingly large number of protein-coding genes generates circular RNAs, these previously overlooked transcripts likely represent key ways that gene functions are expanded and modulated (Kramer, 2015).

Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences; 1) DM1 is associated with a CTG repeat located in the 3' untranslated region of the gene DMPK, and 2) DM2 is associated with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality this study examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. The results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology (Machuca-Tzili, 2006).

The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2

muscleblind (mbl) is involved in terminal differentiation of adult ommatidia. mbl is a nuclear protein expressed late in the embryo in pharyngeal, visceral, and somatic muscles, the ventral nerve cord, and the larval photoreceptor system. All three mbl alleles studied exhibit a lethal phenotype and die as stage 17 embryos or first instar larvae. These larvae are partially paralyzed, show a characteristically contracted abdomen, and lack striation of muscles. Analysis of the somatic musculature shows that the pattern of muscles is established correctly, and they form morphologically normal synapses. Ultrastructural analysis, however, reveals two defects in the terminal differentiation of the muscles: inability to differentiate Z-bands in the sarcomeric apparatus and reduction of extracellular tendon matrix at attachment sites to the epidermis. Failure to differentiate both structures could explain the partial paralysis and contracted abdomen phenotype. Analysis of mbl expression in embryos that are either mutant for Dmef2 or ectopically express Dmef2 places mbl downstream of Dmef2 function in the myogenic differentiation program. mbl, therefore, may act as a critical element in the execution of two Dmef2-dependent processes in the terminal differentiation of muscles (Artero, 1998).

muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins

The embryonic lethal gene muscleblind (mbl) has been isolated as a suppressor of the sev-svp2 eye phenotype. Analysis of clones mutant for mbl during eye development shows that it is autonomously required for photoreceptor differentiation. Mutant cells are recruited into developing ommatidia and initiate neural differentiation, but they fail to properly differentiate as photoreceptors. Molecular analysis reveals that the mbl locus is large and complex, giving rise to multiple different proteins with common 5' sequences but different carboxy termini. Mbl proteins are nuclear and share a Cys3His zinc-finger motif which is also found in the TIS11/NUP475/TTP family of proteins and is highly conserved in vertebrates and invertebrates. Functional analysis of mbl, the observation that it also dominantly suppresses the sE-JunAsp gain-of-function phenotype and the phenotypic similarity to mutants in the photoreceptor-specific glass gene suggest that mbl is a general factor required for photoreceptor differentiation (Begemann, 1997).


REFERENCES

Search PubMed for articles about Drosophila Muscleblind

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Biological Overview

date revised: 26 December 2015

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