Date of Award

Fall 1-1-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy In Regulatory Biology Degree

Department

Biological, Geological and Environmental Sciences

First Advisor

Komar, Anton A.

Second Advisor

Dr. Crystal M. Weyman

Third Advisor

Dr. Barsanjit Mazumder

Abstract

Translation initiation is the rate-limiting and tightly regulated step of protein synthesis. Cap-dependent translation initiation accounts for about 95% of cellular mRNAs. Around 3-5% of cellular mRNAs have been found to contain a cis-regulatory element (IRES) which can recruit ribosomes in a cap-independent manner. IRESs support protein synthesis under cellular stress conditions when cap-dependent translation is inhibited. Differentiation in 23A2 myoblast cells can be induced by culturing cells in serum-free differentiating media (DM). During 23A2 cellular myoblast differentiation, approximately 30% of cells undergo apoptosis as a result of stress caused by serum withdrawal in order to induce differentiation. The expression of the pro-apoptotic Bcl2 family member PUMA was found to be elevated when myoblasts cells were cultured in differentiating media. PUMA was identified as a critical regulator for apoptosis associated with skeletal myoblast differentiation. It was reported earlier that translation of PUMA was found to be regulated during skeletal myoblast differentiation. PUMA was found to be translated by an IRES-mediated mechanism when skeletal myoblast cells were switched from growth media to differentiating media conditions. PUMA was found to be actively translated despite reduced global protein synthesis. PUMA translation was found to proceed by a cap-independent mechanism precluding the need for canonical translation initiation factors like eIF4E and eIF2a. Experimental analyses confirmed the presence of an IRES element located within PUMA 5’UTR. In this study, we attempted vii to understand the mechanism of PUMA internal initiation and identify canonical and noncanonical factors required for PUMA translation. Unlike Hepatitis C viral IRES, the 40S ribosomal subunit is not recruited directly to PUMA IRES during initiation. We also investigated the canonical initiation factors required by PUMA IRES. Using specific inhibitors for eIF4A and eIF4G, we found that PUMA translation requires eIF4A and intact eIF4G for its activity. We further attempted to identify a specific RNA-binding protein which may act as a co-factor and assists PUMA translation under conditions that inhibit cap-dependent translation and activate IRES mediated translation. RNA affinity pull-down assay and mass spectrometric analysis helped us to identify Hsp70 binding to PUMA IRES with high affinity in differentiating media. We confirmed PUMA IRES and Hsp70 protein interaction by gel retardation assay and competition assay. Further, antibody depletion assay confirmed that Hsp70 is an essential factor for PUMA translation.

Download

Included in

Biology Commons

COinS