Juan M. Fernandez-Costa

Drosophila Muscleblind Is Involved in troponin T Alternative Splicing and Apoptosis

Background 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. Methodology/Principal Findings We 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 nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing 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. Conclusions/Significance Taken together our genetic approach 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.

Alternative splicing regulation by Muscleblind proteins: from development to disease

Regulated use of exons in pre‐mRNAs, a process known as alternative splicing, strongly contributes to proteome diversity. Alternative splicing is finely regulated by factors that bind specific sequences within the precursor mRNAs. Members of the Muscleblind (Mbl) family of splicing factors control critical exon use changes during the development of specific tissues, particularly heart and skeletal muscle. Muscleblind homologs are only found in metazoans from Nematoda to mammals. Splicing targets and recognition mechanisms are also conserved through evolution. In this recognition, Muscleblind CCCH‐type zinc finger domains bind to intronic motifs in pre‐mRNA targets in which the protein can either activate or repress splicing of nearby exons, depending on the localization of the binding motifs relative to the regulated alternative exon. In humans, the Muscleblind‐like 1 (MBNL1) proteins play a critical role in hereditary diseases caused by microsatellite expansions, particularly myotonic dystrophy type 1 (DM1), in which depletion of MBNL1 activity through sequestration explains most misregulated alternative splicing events, at least in murine models. Because of the involvement of these proteins in human diseases, further understanding of the molecular mechanisms by which MBNL1 regulates splicing will help design therapies to revert pathological splicing alterations. Here we summarize the most relevant findings on this family of proteins in recent years, focusing on recently described functional motifs, transcriptional regulation of Muscleblind, regulatory activity on splicing, and involvement in human diseases.

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

SUMMARY 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 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, we carried out a genetic screen 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.

Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model.

ABSTRACT Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1) disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor) or muscleblind, or by RNA interference (RNAi)-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis. Summary: Increased apoptosis and autophagy are processes that lead to muscle mass wasting in DM1, which is one of the most debilitating symptoms of the disease.

Myotonic dystrophy: candidate small molecule therapeutics

Myotonic dystrophy type 1 (DM1) is a rare multisystemic neuromuscular disorder caused by expansion of CTG trinucleotide repeats in the noncoding region of the DMPK gene. Mutant DMPK transcripts are toxic and alter gene expression at several levels. Chiefly, the secondary structure formed by CUGs has a strong propensity to capture and retain proteins, like those of the muscleblind-like (MBNL) family. Sequestered MBNL proteins cannot then fulfill their normal functions. Many therapeutic approaches have been explored to reverse these pathological consequences. Here, we review the myriad of small molecules that have been proposed for DM1, including examples obtained from computational rational design, HTS, drug repurposing, and therapeutic gene modulation.

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 we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe the conservation of the sequence motif KRAEK in isoforms C and E and propose 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 β-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.

Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

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. Here we identify two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. We also show 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 resulted in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies had improved muscle function in climbing and flight assays, and had longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3’ untranslated region of the DMPK gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, miR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652 all failed to show consistent differences in serum expression in subsequent validation experiments.

A GFP-tagged muscleblind C protein isoform reporter construct.

Drosophila muscleblind (mbl), the ortholog of human Muscleblind-like 1 (MBNL1) gene involved in Myotonic Dystrophy (DM), gives raise to protein isoforms MblA to G. The specific functions and subcellular distribution of isoforms are still largely unknown. To overcome the lack of isoform-specific antibodies we generated transgenic flies that express a GFP:MblC fusion protein under the control of the Gal4/UAS system. The reporter fusion protein was able to functionally complement mbl loss of function mutations, demonstrating activity, and accumulated predominantly in adult muscle nuclei. The fluorescent nature of the reporter makes it appropriate for live imaging detection of MblC protein isoform.