Date of Award

6-1-2014

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology/Molecular, Cellular, and Organismal Biology

First Advisor

Kenneth Kleene

Second Advisor

Rick Kesseli

Third Advisor

Linda Huang

Abstract

Spermatogenesis is the process by which diploid stem cells differentiate into haploid male gametes, spermatozoa. As haploid cells differentiate into spermatozoa, they undergo profound changes in morphology and physiology, including total reorganization of chromatin in the nucleus which results in the inability to synthesize new mRNAs about one week before the differentiation of sperm is complete. Consequently, many mRNAs, such as the protamine 1 (Prm1) mRNA and sperm mitochondria-associated cysteine rich-protein (Smcp) mRNA, are transcribed in early haploid spermatogenic cells and stored as translationally inactive messenger ribonucleoprotein particles (free-mRNPs) for several days to a week before the mRNA is translated to make protein in late haploid spermatogenic cells. The initial translational repression is critical for normal sperm development, since premature activation of translation leads to deformed spermatozoa and male subfertility or infertility. The mechanisms that regulate mRNA translation in haploid spermatogenic cells are poorly understood. My research explores the mechanisms of translational regulation of Prm1 and Smcp mRNAs. The goal of my research is to identify factors and elements that repress the translation of Prm1 and Smcp mRNAs in round spermatids and activate their translation in late spermatids.

Prm1 and Smcp mRNA are the only two mRNAs extensively studied through mutations in transgenic mice. Mutation in transgenic mice is the best method of identifying cis-elements in spermatogenic cells. Previous studies of mutations in transgenic mice have identified cis-elements which are necessary to repress the Prm1 mRNA and Smcp mRNAs in transgenic mice. Using UV-crosslinking RNA binding assays, RNA affinity chromatography and mass spectrometry sequencing, my research demonstrates that Y-box protein 2 (YBX2) binds both elements suggesting that YBX2 is an important mRNA specific translational repressor. UV-crosslinking assays also reveal that YBX2 is less specific in its binding specificity than was previously known, implying that YBX2 represses many mRNAs. My research also demonstrates that proper repression of the Smcp mRNA in early haploid cells requires interactions between multiple elements in the 5'UTR and the 3'UTR.

Chowdhury -- Additional data for Chapter 2.pdf (653 kB)
Supplemental Figures, Additional Data for Chapter 2

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