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RNA binding proteins (RBPs) have emerged as important regulators of gene expression. RBPs typically contain RNA binding domains that recognize a specific sequence and/or structural motifs within the RNA. This allows them to modulate metabolism of RNAs in several possible ways including regulation of alternative splicing and processing, polyadenylation, translocation, localization, modification, stability, or translation. Previous studies have shown the Musashi (MSI) RBP family to be highly expressed in the retina, and more specifically, photoreceptors, but the importance of this expression remains largely unknown. We identified the MSI proteins as potential regulators of alternative exon splicing in murine photoreceptors. We hypothesized that the MSI proteins are essential splicing factors needed to produce photoreceptor-specific transcripts and that inactivation of the Msi genes would lead to decreased photoreceptor function and survival subsequent to aberrant splicing. We also predicted that the MSI proteins were regulating splicing of transcripts involved in ciliogenesis and outer segment morphogenesis. To test our hypothesis, I generated Cre-LoxP conditional knockout mice to inactivate the Msi genes either in the entire retina and ventral forebrain or specifically rod photoreceptors. I found that both rod and cone photoreceptor function was completely absent after pan-retinal inactivation of both Msi genes. I also discovered alterations in retinal progenitor cell proliferation and decreased retinal cell survival at later ages in the absence of the MSI proteins. When analyzing the morphology of the outer segment and connecting cilium in the absence of MSI, I found defects only in outer segment morphology. Furthermore, I found that the MSI proteins regulate the photoreceptor-specific splicing of several outer segment and cilia-related transcripts including Bbs8, Cc2d2a, Cep290, and Prom1. Lastly, we found that deletion of these photoreceptor-specific exons in C57BL6/J mice did not significantly affect photoreceptor function.

Alternative pre-mRNA splicing provides an important mechanism for generating the diverse array of proteins required to generate complex tissue and cell types from a limited genome. Therefore, the proper regulation of alternative splicing is vital to shape cellular identity and function. As consequence, defects in alternative splicing are associated with disease phenotypes that can range from systemic syndromes to the dysfunction of single cell types. For example, heterozygous mutations in ubiquitously expressed components of the spliceosome lead to photoreceptor specific cell death. This suggests that the topography of alternative splicing in photoreceptors cells may be unique, a notion which is supported by reports of photoreceptor specific splicing events. However, the mechanisms mediating photoreceptor specific splicing, and the reason why photoreceptors are uniquely sensitive to perturbations in the splicing machinery, remain unknown. In this work, I characterize the alternative splicing program of photoreceptor cells using exon 2A of the BBS8 gene as a model. The photoreceptor specific exon 2A was recently discovered through a mutation in the 3’ splice site that was linked with non-syndromic retinitis pigmentosa (RP). Skipping of this exon in photoreceptor cells was thought to limit the phenotype of the mutation to RP, rather than the systemic disease Bardet-Biedl syndrome (BBS). I show that the IVS1-2A>G mutation in BBS8 leads to missplicing of exon 2A, producing a shift in the reading frame predicted to eliminate the BBS8 protein specifically in photoreceptor cells. I also show that in the absence of splicing elements within the exon, the splicing of exon 2A is directed entirely by sequences located within the adjacent introns. To gain a more expansive view of the photoreceptor splicing program, I utilize mouse models to isolate the gene expression and alternative splicing profile of photoreceptor cells by RNA sequencing. Bioinformatics analysis indicates that while photoreceptors share a general splicing pattern with other neurons, they exhibit a distinct program that affects a broad set of genes. Cell type specific splicing in photoreceptors appears to be regulated by a combinatorial mechanism which involves activation by the Musashi proteins in the absence of many typical neuronal splicing regulators. This program controls a subset of exons, including BBS8 exon 2A, which are spliced in a “switch-like” manner to produce photoreceptor specific protein isoforms. These splicing events share a temporal inclusion pattern which precedes the development of the light sensing outer segment. Remarkably, multiple switch-like exons are located within genes that are necessary for the biogenesis and maintenance of primary cilia. This suggests that alternative splicing may modulate protein function to allow for development and maintenance of the unique structure of photoreceptor cells. This work provides a foundation on which to characterize the regulation of alternative splicing in photoreceptor cells, and identifies multiple splicing events which may impact the function of the photoreceptor outer segment.