Exploring the functional conservation of muscleblind (Mbl) proteins

Abstract

Muscleblind (Mbl) is an evolutionarily conserved family of proteins involved in many aspects of RNA metabolism, including alternative splicing. Disruption of Muscleblind in several animals lends to a variety of defects and disease, including the multi-systemic disorder Myotonic Dystrophy (DM). Though much is known about the involvement of Muscleblind in DM, there is much basic knowledge of the protein's function to be discovered. We approach this problem by exploring the functional conservation of a diverse subset of Muscleblind homologs. The functions of Muscleblinds from a basal metazoan, Trichoplax adhaerens, a primitive chordate, Ciona intestinalis, and the model organisms, Drosophila melanogaster and Caenorhabditis elegans were compared to human Muscleblind-like (MBNL). The zinc finger RNA-binding domains are the most conserved region between homologs, suggesting a conserved role in RNA binding and splicing regulation. To test this, we used splicing reporter assays with validated human MBNL-regulated mini-genes and performed RNA sequencing experiments in mouse embryonic fibroblasts (MEFs). Additionally, we accessed the subcellular localization of the homologs to determine conservation of extra-nuclear functions. Reporter assays in HeLa cells showed that the homologs can positively and negatively regulate splicing. Our RNA-seq experiments led us to discover hundreds of endogenously regulated splicing events, including the identity of the transcripts, direction of splicing regulation, types of splicing events, and the magnitude of alternate exon inclusion in the spliced mRNAs. Additionally, we identified a spectrum of splicing events, from those uniquely regulated by a single Muscleblind, to events regulated by all Muscleblinds, and, characterized the variation in splicing activity that exists between homologs. A subset of events regulated by mammalian Muscleblind were oppositely regulated by non-mammalian homologs. Muscleblinds show nuclear-cytoplasmic localization, which suggests conservation in extra-nuclear functions. In conjunction with exon and intron sequences, this information provides a future tool to discover conserved and novel RNA regulatory elements used by diverse Muscleblinds to regulate splicing and in putative cytoplasmic functions. These data could also be used to determine functionally important residues in Muscleblind proteins and help us better understand the protein family.