Talk abstracts

Talk on Saturday 08:30-08:45am submitted by Pieter Spealman

Alternative translation initiation site usage under environmental stress conditions.

Pieter Spealman (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Cells respond to changes in the environment through coordinated changes in gene expression. Recent work suggests the cellular response to stress involves a shift ribosome initiation preference from AUG translation initiation sites (TISs) to non-AUG, alternative TISs (1). The use of alternative TISs could have wide-ranging effects on gene expression through the generation of novel N-terminal extension protein isoforms. These isoforms qualitatively differ from the canonical type by the inclusion of additional peptides at the N-terminus (2). Examples of N-terminal isoforms have been shown to change protein functions by affecting transcription factor binding targets, subcellular location of enzymes, and altering protein degradation rates.
Ribosome profiling is a deep-sequencing method that reveals the location of translating ribosomes on mRNA (3) and has been used to identify dozens of putative N-terminal extensions in Saccharomyces cerevisiae (4), mouse, and human (5). While this work suggests that these isoforms could play an important role in the stress response, only a small number have been shown to be functional. Here, we use conservation as a proxy for function and compare isoform expression in two species of budding yeast under multiple environmental conditions using ribosome profiling (6). We identify 131 N-terminal isoforms between the two species, 124 of which are condition specific, and 116 of which had not been previously described. Using mass spectrometry data we identified 51 genes with N-terminal isoforms present at the level of the proteome (7). While we find evidence of conservation and purifying selection acting upon N-terminal extensions only 6 genes are observed to have isoforms in both species, suggesting that function may be conserved while condition specificity has diverged. To further evaluate condition specific expression of N-terminal extensions we are constructing a fluorescent reporter assay.

1. Botao L. et al. Wiley Interdiscip Rev. RNA (2014)
2. Michel, A. M. et al. Wiley Interdiscip Rev RNA 4, 473–90 (2013).
3. Ingolia, N. T. et al. Science 324, 218–23 (2009)
4. Gerashchenko, M. V. et al. Proc. Natl. Acad. Sci. U.S.A. 109, 17394–9 (2012).
5. Van Damme, P. et al. Mol. Cell Proteomics 13,1245–61 (2014).
6. Holcik, M. et al. Nat. Rev. Mol. Cell Biol. 6, 318–27 (2005).
7. Iesmantavicius, V. et al. Mol. Cell Proteomics 13, 1979–92 (2014).

Keywords: Translation Regulation, Gene Expression, Stress Response