Date of Award

12-31-2014

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology/Molecular, Cellular, and Organismal Biology

First Advisor

Kenneth C. Kleene

Second Advisor

Rick Kesseli

Third Advisor

Alexey Veraksa

Abstract

The sperm-mitochondria-associated cysteine-rich protein (SMCP) is a male germ cell-specific protein that localizes to the outer membranes of sperm mitochondria and increases sperm motility. The Smcp mRNA is transcribed in early spermatids, and stored in a translationally repressed state for ~7 days before translation is activated in late spermatids. Identifying the cis-elements and trans-factors that repress the Smcp mRNA in early spermatids is important because these factors and elements coordinate the translational activity of hundreds of mRNAs.

A mutation was studied in transgenic mice in which the 16 nucleotides downstream of the first poly(A) signal in the Smcp 3'UTR were replaced with the 17 nucleotides downstream of the poly(A) signal from the pEGFP plasmid 3'UTR. Replacing this sequence of the Smcp 3'UTR eliminates two elements that are conserved in many mammalian Smcp mRNAs. My research using the GFP reporter and analysis of polysomal loading demonstrates that the mutation eliminates repression of a Smcp-Gfp transgenic mRNA in early spermatids.

Studies in our lab demonstrate that Y-box protein 2 (YBX2) binds the 3' termini of the protamine 1 (Prm1) and Smcp 3'UTRs, which have been demonstrated with mutations in transgenic mice to be necessary for repression in early spermatids. My research demonstrates that depletion of YBX2 in Ybx2-null mice eliminates the translational repression of the Smcp and Prm1 mRNAs in early spermatids.

The localization of the Smcp mRNA in spermatids was studied using RNA-fluorescent in situ hybridization (RNA-FISH). The Smcp mRNA probe detected a signal in a germ cell-specific granule called the chromatoid body. It has been speculated that the chromatoid body stores repressed mRNAs in early spermatids. My RNA-FISH studies reveal that translationally repressed and translationally active mRNAs are concentrated in the chromatoid body implying that localization is independent of translational activity. A probe for the Smcp intron also localized to the chromatoid body suggesting that the Smcp pre-mRNA may be spliced in the chromatoid body. This is the first demonstration with RNA-FISH that translationally active mRNAs and introns localize to the chromatoid body. This research has permitted the formulation of a speculative model of translational repression of the Smcp mRNA.

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