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Translational Control in Fungi

  • Author(s): Clarkson, Bryan Kensey
  • Advisor(s): Doudna, Jennifer A
  • et al.
Abstract

Regulation of protein synthesis is critical for coordinating cellular activity with environmental cues. The extensive discrepancy between messenger RNA (mRNA) and protein levels exemplifies the pervasiveness of translational control. Accordingly, dysregulation of translation is associated with diseases, ranging from cancer to developmental, neuropsychiatric and metabolic disorders. Understanding cellular adaptation and the molecular underpinnings of diverse diseases requires a detailed understanding of translational control mechanisms. This work discusses the contributions of an essential translation factor to genome-wide protein synthesis and the ability of an unusual RNA structure element from Hepatitis C virus to promote translation in fungi.

Eukaryotic initiation factor 4G (eIF4G) is essential for protein synthesis. It simultaneously interacts with message- and ribosome-associated factors to help recruit and position the ribosome on the mRNA. The genomes of a wide variety of eukaryotes encode multiple eIF4G isoforms, some of which have unique translational functionality. To test the hypothesis that the two different individual eIF4G isoforms in yeast make distinct contributions to translation, I examined the effect of removing each eIF4G isoform on growth and translation in Saccharomyces cerevisiae. Strains expressing a single isoform at wild-type levels revealed that both isoforms have the capacity to support wild-type growth and translation rates. In fact, no messages expressed under standard laboratory conditions rely strongly on a particular eIF4G isoform for their translation. Environmental fluctuations affect the fitness of wild-type and isoform-specific strains equally, suggesting a large functional overlap exists between yeast eIF4G isoforms. Therefore, encoding eIF4G proteins from distinct genetic loci ensures maintenance of adequate eIF4G levels, which is critical for cellular fitness.

Several stresses reduce eIF4G levels to regulate translation. To determine whether there are message-specific effects of eIF4G reduction I compared the translational efficiency of all yeast mRNAs under wild type and eIF4G-limiting conditions. The translation of genes that encode messages with longer poly-adenosine (poly(A)) tails are preferentially sensitive to eIF4G limitation. To test the hypothesis that translational enhancement by the poly(A) tail requires adequate eIF4G, I determined the effects of limiting eIF4G on the translation of mRNAs with varying poly(A) tail lengths. eIF4G limitation does not alter the relationship between poly(A) tail length and translation, in vitro. Therefore, mRNA-specific differences in the requirement for eIF4G are mediated by a poly(A)-independent mechanism.

The Hepatitis C virus (HCV) is a global public health threat and new therapeutics are needed. The development of new treatments requires a mechanistic understanding of critical stages in the viral life cycle. HCV utilizes an internal ribosome entry site (IRES) to initiate the synthesis of proteins required for viral replication. Extensive biochemical and structural studies have assigned functions to IRES components, however the influence of host factors on IRES activity remains obscure. To establish a genetic system for the study of host factor influence we determined the ability of the HCV IRES to initiate translation in Schizosaccharomyces pombe. The HCV IRES does not efficiently promote translation in S. pombe in vivo or in vitro. Initial steps have been taken to identify mammalian factors that are able to stimulate HCV IRES-mediated translation in S. pombe.

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