Biochemistry, Department of

 

Date of this Version

4-2014

Citation

Gerashchenko M. Redox regulation of protein translation in eukaryotes. (2014) Ph.D. diss., University of Nebraska-Lincoln.

Comments

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professors Dmitri E. Fomenko and Vadim N. Gladyshev. Lincoln, Nebraska: April, 2014

Copyright (c) 2014 Maxim Gerashchenko

Abstract

Gene expression may be controlled at multiple levels, e.g., through genomic architecture, transcription and translation. In the current work, we focused on regulation of protein synthesis. Historically, the investigation of the regulation of gene expression at the level of translation lagged behind the transcriptional control because of the lack of accessible high-throughput methods. Our research has begun with the finding of the use of alternative non-AUG start codon in thioredoxin-glutathione reductase (TGR), a selenoprotein involved in redox control during male reproduction. The use of this codon, CUG, relies on the Kozak consensus sequence and ribosomal scanning mechanism. However, the CUG serves as an inefficient start codon that allows downstream in-frame initiation, generating two isoforms of the enzyme in vivo and in vitro from the same mRNA. These findings were extended with the use of systemic, proteome-wide approaches, that supported targeted discovery of initiation start sites. For this purpose, a new technology, ribosomal profiling, was employed. It embraced high-throughput sequencing and offered analyses of ribosome occupancy along the mRNA at a single nucleotide resolution. We applied this technique to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Oxidative stress elicited by hydrogen peroxide led to a massive and rapid increase in ribosome occupancy of short upstream open reading frames (uORFs), including those with non-AUG translational starts, and N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially duration of the elongation step. We identified proteins whose synthesis was rapidly regulated by hydrogen peroxide post-transcriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. Nevertheless, a number of proteins were regulated post-transcriptionally even at the 5 min time point. These data defined the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggested that "potentiation" (co-regulation of the transcript level and translation) is a feature of oxidative stress. Finally, we expanded this research to better define conditions for ribosome profiling, which are broadly applicable for studies on regulation of translation.

Advisors: Vadim N. Gladyshev and Dmitri E. Fomenko