TCP-seq records the complete cycle of mRNA-ribosome interactions <em>in vivo</em> — ASN Events

TCP-seq records the complete cycle of mRNA-ribosome interactions in vivo (#34)

Nikolay E. Shirokikh 1 2 , Stuart K. Archer 3 , Traude H. Beilharz 4 , David Powell 3 , Ross Hannan 5 , Thomas Preiss 1 6
  1. Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  2. State Socio-Humanitarian University, Kolomna, Moscow region, Russia
  3. Monash Bioinformatics Platform, Monash University, Melbourne, VIC, Australia
  4. Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  5. Department of Cancer Biology & Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  6. Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

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 consists of dynamic interactions between eukaryotic initiation factors (eIFs) and the small ribosomal subunit (SSU) and concludes with joining of the large ribosomal subunit (LSU) to form a full ribosome at the start codons of mRNA. However, no methods existed to study the mechanistic intricacies and regulation of translation initiation transcriptome-wide.

To address this, we developed translation complex profile sequencing (TCP-seq)1, a method related to the ribosome profiling approach. TCP-seq is uniquely capable of recording positions of any type of ribosome-mRNA complex across all RNA. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into full ribosome and SSU fractions. Ribosome and SSU-derived footprints are then deep sequenced separately, generating near-native complex distribution profiles during the initiation, elongation, termination and recycling stages of translation.

TCP-seq use was highly successful in discerning long-standing mechanistic questions of translation in yeast model system, including resolving the elusive ‘scanning’ process, visualizing start codon recognition events and capturing diverse conformations of elongating ribosomes1. Here we outline further improvements to TCP-seq that make it a versatile, flexible approach which can be used to study regulation of protein synthesis with unprecedented depth of detail. Combined with yeast strains with altered activity of eIFs, it can be used to resolve differential initiation mechanisms utilised across transcripts and pinpoint regulatory elements in 5'UTRs of mRNA. Used in combination with affinity binding (co-immunoprecipitation), TCP-seq can provide a snapshot of all translation events that involve a certain protein factor and/or cellular location. Adapted to perform on mammalian cells, TCP-seq can be used to identify mechanisms of protein synthesis control active in biologically important microenvironments, such as nutrient deficiency, ageing and development of cancer, and thus provide entirely new perspectives on the development of better treatments.

  1. Archer, S.K., Shirokikh, N.E., Beilharz, T.H., Preiss, T. Dynamics of ribosome scanning and recycling revealed by translation complex profiling. Nature. 2016 535, 570-574. PMID: 27437580 doi: 10.1038/nature18647
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