Poster abstracts

Poster number 143 submitted by Pieter Spealman

Comparative translatomics reveals a class of conserved, stress responsive, non-canonical uORFs

Pieter Spealman (Department of Biological Sciences, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213. ), Armaghan W. Naik, Robert F. Murphy (Computational Biology Department, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213.), Scott Kuersten, Lindsay Freeberg (Illumina Inc., Madison, WI, USA ), Gemma May, C. Joel McManus (Department of Biological Sciences, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213. )

Abstract:
Upstream open reading frames (uORFs) have emerged as major cis-acting elements involved in regulating mRNA turnover and translation (1,2). While the most well-known uORFs initiate using the canonical AUG start codons, an ever increasing number of uORFs are being proposed that initiate at near-cognate codons (NCCs) (3,4). While translation of such NCC uORFs increases under stress (5), their function and evolution remain largely uncharacterized.
We used comparative translatomics to investigate AUG and NCC uORFs in three yeast species. This required mapping both major and minor transcription start sites (TSSs) in S. cerevisiae, S. paradoxus, and S. uvarum. Consistent with prior work, AUG triplets were depleted from the TLS in each species. Surprisingly, we found the majority of NCC codons to be both enriched in the TLS and conserved between species, unlike AUG. This is consistent with the model that NCC triplets are under selective pressure to maintain translation initiation at uORFs. We then searched the TLs for functional uORFs using a novel machine learning algorithm (uORF-seqr) and ribosome profiling data from each species. Our analysis identified both conserved and species-specific AUG- and NCC-uORFs in hundreds of orthologous TLSs. Numerous features, such as RNA-binding protein distribution, the rate of uORF inclusion in transcript isoforms, and the effect each uORF type has on the translation of the downstream gene, suggest these AUG and NCC-uORFs represent distinct regulatory elements. Because NCCs may be preferentially translated under stress, we also performed ribosome profiling in S. cerevisiae and S. paradoxus after acute amino-acid starvation. For both species, the relative AUG-uORF usage decreased under stress, while NCC usage increased dramatically. Notably, several genes with conserved stress-responsive NCC-uORFs are central to transcription and translation. These results suggest that NCC-uORFs may play an important role in the regulation of mRNA levels and translation in response to stress, discrete from that observed for AUG-uORFs.

References:
1. Wethmar, K. The regulatory potential of upstream open reading frames in eukaryotic gene expression. Wiley Interdiscip Rev Rna (2014).
2. Ingolia et al. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell (2014).
3. Zhang et al. An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res. (2011).
4. Brar, G. et al. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science (2011).
5. Ingolia et al. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. (2009)

Keywords: upstream open reading frames, translation regulation, gene expression