Poster Presentation 39th Annual Lorne Genome Conference 2018

Dissecting the roles of eIF2 and eIF3 during translation using TCP-seq   (#270)

Susan Wagner 1 , Stuart K Archer 2 , Nikolay E Shirokikh 1 , Neelam Sen 3 , Nick T Ingolia 4 , Alan G Hinnebusch 3 , Thomas Preiss 1 5 , Leos S Valasek 6
  1. The John Curtin School of Medical Research, The Australian National University, Acton, ACT, Australia
  2. Monash Bioinformatics Platform, Monash University, Melbourne, Vic, Australia
  3. Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
  4. Department of Molecular and Cell Biology, Center for RNA Systems Biology, University of California, Berkeley, California, USA
  5. Victor Chang Cardiac Research Institute, Sydney, NSW, Australia
  6. Institute of Microbiology, Czech Academy of Sciences, Prague, The Czech Republic

Regulation of mRNA translation is a key element of eukaryotic gene expression control and, for example, prominently involved in adjusting the proteome to rapid changes in the cellular environment. The most highly regulated phase of translation is the initiation phase, during which ribosomes are recruited to the mRNA 5’ untranslated region (UTR). Over a dozen eukaryotic initiation factors (eIFs) assist the small ribosomal subunit (SSU) in binding near the 5’ cap, scan the 5’ UTR, recognise the appropriate start codon and unite with the large ribosomal subunit (LSU). Two key factors involved in these processes are eIF3, a multifunctional factor that wraps around much of the SSU, and eIF2, which forms a complex with initiator tRNA and is pivotal in start codon recognition. The prevalence of upstream open reading frames and near-cognate start codons among mRNA makes start codon recognition an important target for quantitative and qualitative control of translation.

In the present study, we use translation complex profile sequencing (TCP-seq) in combination with affinity purification of eIF-SSU complexes to investigate the dynamic involvement of eIF2 and eIF3 in different sub-steps of translation. We focus on the regulatory role of upstream open reading frames (uORFs) and the mechanism of resumption of scanning and reinitiation downstream. We also study aspects of start codon recognition that relate to the poorly understood role of the immediate sequence context surrounding efficient start codons– the ‘Kozak context’. We identify translation initiation complexes that differ in their composition and functionality during start codon recognition.