Author ORCID Identifier

https://orcid.org/0000-0002-0160-3757

Semester

Fall

Date of Graduation

2022

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Peter Stoilov

Committee Co-Chair

Visvanathan Ramamurthy

Committee Member

Visvanathan Ramamurthy

Committee Member

Maxim Sokolov

Committee Member

Roberta Leonardi

Committee Member

Ariel Agmon

Abstract

The Musashi (Msi) family of RNA binding proteins consists of two paralogs, Msi1 and Msi2, that are highly conserved across species. The two paralogs have emerged as factors that promote stem cell proliferation by post-transcriptionally regulating translation. In addition to their expression in stem cells, the Musashi proteins are also expressed in postmitotic neurons, including the photoreceptor cells. The Musashi proteins have been observed to maintain high expression levels in the postmitotic photoreceptors within the eye of both invertebrates and vertebrates. These observations suggest an additional role in the maintenance of terminally differentiated neurons.

Building upon these observations, we investigated the role of Musashi individually and in combination in mature photoreceptors. Using a tamoxifen-inducible mouse model, I generated single and combined deletion of Msi1 and Msi2 in mature photoreceptor cells. Our results show that the Musashi proteins are required for the function and viability of mature photoreceptors. Global analysis of the Msi1 targets in the retina showed binding to UAG motifs predominantly located in introns and 3’-UTRs. Using RNA-sequencing and proteomics analysis, with the incorporation of the publicly available single-cell RNA seq, we found that in mature photoreceptors, the Musashi enhance the expression of proteins in high demand. Among these targets are proteins needed for the daily regeneration of the light sensory organelle of the photoreceptors. Collectively, the data provide new insights on the targets, possible molecular mechanisms, and function of the Musashi in mature photoreceptors. The results support a model of the Musashi proteins acting as a posttranscriptional activator for protein expression in mature photoreceptors.

In the course of our work, an unusual behavior of the 13A4 antibody to prominin-1 (Prom1) prompted us to analyze its epitope. Prom1 is a transmembrane protein with a role in the morphogenesis of photoreceptor outer segment disk membranes. Mutations in the Prom1 gene have resulted in various forms of retinal degeneration affecting rods and cones. Scanning deletion mutagenesis and structural modeling demonstrated that mAB 13A4 recognizes a structural epitope that is affected by the inclusion of the alternative exon 19 during photoreceptor maturation. Consequently, the reactivity of mAB 13A4 towards the photoreceptor specific isoform of PROM1 is significantly reduced on a Western blot leading to gross underestimation of PROM1 protein levels in the retina.

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