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.     

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. Here we report 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. Our results reveal that Mbl recognizes complex RNA secondary structures.     

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.     

Modifications to toxic CUG RNAs induce structural stability,rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model

CUG repeat expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein–RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2′-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2′-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.     

Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex

To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1(CUG)), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1(CUG) complexes from normal myoblasts. In normal myoblasts, MBNL1(CUG) bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1(CUG) colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1(CUG) complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1(CUG) complexes results from aberrant protein synthesis or stability and is unlinked to PKCα function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1(CUG) complexes to allow both the reinforcement and expansion of RNA processing defects.     

Conservation of context-dependent splicing activity in distant Muscleblind homologs

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.     

Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n > 50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscleblind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.     

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. Here we demonstrate 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.