Nikolay Shirokikh, Department of Genome Sciences, JCSMR was invited to present in the Plenary Session: "Translation" as part of the 21st Annual Meeting of the RNA Society, in Kyoto this weekend. Nikolay spoke about work that has been carried out by a team of researchers from JCSMR, Monash Unviersity and the Moscow Regional State Institute of Humanities and Social Studies (pictured). The full abstract of Niokolay's presentation is available below:
Dynamics of ribosome scanning and recycling revealed by translation complex profile sequencing.
Stuart Archer, Nikolay Shirokikh, Traude Beilharz and Thomas Preiss
Regulation of mRNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated initiation factors (eIFs) near the mRNA 5' cap; the SSU then 'scans' in the 3' direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome (RS). Scanning and other dynamic aspects of the initiation model remain conjecture as methods to trap early intermediates are lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-Seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5'UTRs. Scanning SSU have 5'-extended footprints (up to ~75 nt), indicative of additional interactions with mRNA emerging from the exit channel, enforcing forward movement. We visualise changes in initiation complex conformation as SSU footprints coalesce into three major sizes at start codons (19, 29 and 37 nt). These share the same 5' start but differ at the 3' end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-Seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.