Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Robin Dowell

Second Advisor

Rui Yi

Third Advisor

Tom Blumenthal

Fourth Advisor

Joaquin Espinosa

Fifth Advisor

Norm Pace

Abstract

The Musashi (Msi) family of RNA-binding proteins is post-transcriptional regulators of gene expression. They were discovered in 1994 as being required for Drosophila sensory organ development. Since then, Msi proteins have been found to enhance cell proliferation and maintain stem cell identities in a multitude of mammalian tissues. In addition, overexpression of Msi proteins is often observed in many types of human cancers, most prominently the widely expressed Msi family member, Musashi-2 (Msi2). Msi2 plays oncogenic roles in hematopoietic, neural, and gastrointestinal tissues. However, Msi2 has received little attention in other tissues in which it is expressed, such as in stratified epithelium. Additionally, the fundamental questions concerning Msi2 such as target recognition, molecular mechanisms of Msi2-mediated gene regulation and cell type-specific functions are poorly understood.

To understand the functions of Msi2 in mouse skin, I identified Msi2 targets using High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in keratinocytes, which are basal progenitor cells that express high levels of Msi2. Analysis of these data revealed a strong preference of Msi2 for 3’UTR binding at regions enriched for a trinucleotide UAG core motif. These data were used in conjunction with RNA-seq and ribosome profiling data in Msi2-depleted keratinocytes and Msi2-normal keratinocytes to reveal a possible mechanism of target regulation. I found that loss of Msi2 increased RNA stability without altering translational efficiency, consistent with Msi2’s role as an inhibitor of gene expression. From these datasets, I generated a list of high confidence, Msi2-regulated targets in keratinocytes. Among these Msi2 targets, I found that genes involved in the regulation of proliferation, cell survival and migration were enriched. Indeed, the Msi2-depleted keratinocytes showed reduced levels of cell proliferation and survival, but increased migration when compared to the keratinocytes with normal levels of Msi2. These results are consistent with observations in wounded skin, where migrating keratinocytes proximal to the wound showed reduced levels of Msi2 in contrast to the normal levels observed in normal keratinocytes distal to the wounded skin. Collectively, these data provide new insights into the targets, molecular mechanisms, and functions of Msi2, and identify an unexpected function of Msi2 in restricting epithelial cell migration in keratinocytes.

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