The Musashi family of RNA-binding proteins is known for its role in stem-cell renewal and are negative regulators of cell differentiation. Interestingly, in the retina, the Musashi proteins MSI1 and MSI2 are differentially expressed throughout the cycle of retinal development, with MSI2 protein displaying robust expression in the adult retinal tissue. In this study, we investigated the importance of Musashi proteins in the development and function of photoreceptor neurons in the retina. We generated a pan-retinal and rod photoreceptor neuron-specific conditional knockout mouse lacking MSI1 and MSI2. Independent of sex, photoreceptor neurons with simultaneous deletion of Msi1 and Msi2 were unable to respond to light and displayed severely disrupted photoreceptor outer segment morphology and ciliary defects. Mice lacking MSI1 and MSI2 in the retina exhibited neuronal degeneration, with complete loss of photoreceptors within six months. In concordance with our earlier studies that proposed a role for Musashi proteins in regulating alternative splicing, the loss of MSI1 and MSI2 prevented the use of photoreceptor-specific exons in transcripts critical for OS morphogenesis, ciliogenesis and synaptic transmission. Overall, we demonstrate a critical role for Musashi proteins in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in stark contrast with the canonical function of these two proteins in the maintenance and renewal of stem cells.

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

23We previously proposed a role for the Musashi proteins, MSI1 and MSI2, in 24 photoreceptor cell development that is mediated by their ability to control alternative splicing. 25 Photoreceptors with simultaneous deletion of Msi1 and Msi2 did not respond to light, displayed 26 severely disrupted OS morphology and axonemal defects. At postnatal day 5, we observed an 27 increase in proliferating retinal progenitor cells in the knockout animals, suggesting delay in 28 photoreceptor development. The loss of Musashi prevented the use of photoreceptor-specific 29 exons in transcripts important for OS morphogenesis, ciliogenesis and synaptic transmission.30 However, deletion of the photoreceptor-specific exons in Ttc8, Cc2d2a, Cep290, Cacna2d4, and 31 Slc17a7 did not impair retinal development or visual function. We demonstrate a critical role for 32 Musashi in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in 33 stark contrast with the canonical function of the two proteins in maintenance and renewal of stem 34 cells. 35 36 37 38 42Alternative splicing is particularly prevalent in vertebrate neurons and is critical for the 43 development and function of vertebrate nervous systems 2-7 . 44 We previously showed that photoreceptor neurons exploit a unique splicing program 8 . 45Motif enrichment analysis suggested that Musashi-1 (MSI1) and Musashi-2 (MSI2), promote the 46 use of photoreceptor specific exons 8 . We further showed that MSI1 is critical for utilization of 47 photoreceptor specific exon in Tetratricopeptide repeat domain protein-8 (Ttc8) 8 . In addition, 48 Musashi promotes the splicing of several photoreceptor specific exons when over-expressed in 49 cultured cells 8 . Recently, analysis of a comprehensive gene expression data set that spanned 50 multiple tissues and cell types from mice and human proved that photoreceptors utilize a unique 51 set of alternative exons that are primarily regulated by MSI1 and MSI2 9 . Furthermore, the work 52 by Ling et. al. demonstrated that Msi1 transcript levels are upregulated in the developing rod 53 photoreceptors and reach exceptionally high levels compared to all other cell types or tissues in 54 the data set. 55 The MSI1 and MSI2 proteins have two highly conserved RNA binding domains (RBDs) 56 in the N-terminal region which show close to 90% sequence identity and recognize a similar 57 UAG motif in RNA 10 . The two RBDs of MSI1 and MSI2 are followed by a less conserved C-58 terminal region which shows approximately 70% sequence identity 11 . The high degree of 59 sequence identity between the MSI1 and MSI2 results in functional redundancy between the two 60 proteins 12,13 . The canonical function of the Musashi proteins is to control mRNA translation in 61 4 the cytosol 14,15 , where they can either block or enhance translation of mRNA depending on 62 cellular context 16-21 . 63 Vertebrate photoreceptors are neurons specialized in detecting and transducing light 64 stimuli. Photoreceptors are characterized by segmented morp...

Alternative splicing in vertebrate photoreceptors and mechanisms underlying retinitis pigmentosa Jesse Sundar 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. I also analyzed mechanisms underlying neurodegeneration in a model of retinitis pigmentosa called the retinal degeneration-10 (rd10) mouse model, which harbors a mutation in the Pde6b gene. In this model, significant photoreceptor degeneration is observed in mice reared under normal light conditions yet rearing these mice in complete darkness significantly increases photoreceptor survival. We hypothesized that the phototransduction cascade was signaling photoreceptor cell death in rd10 mice. To test the hypothesis, I inactivated either transducin or rhodopsin signaling in rd10 mice. I found that inactivating transducin signaling in rd10 mice did not protect against light-dependent photoreceptor cell death whereas inactivation of rhodopsin signaling in rd10 mice did protect against light-dependent photoreceptor cell death. I also found that there was a significant reduction in the fu...

No abstract