title: Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions during translation initiation, elongation and termination.
abstract: Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.
authors.affiliation: EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.|State Socio-Humanitarian University, Kolomna, Russia.
authors.forename: Nikolay E
authors.initials: NE
authors.lastname: Shirokikh
authors.affiliation: Monash Bioinformatics Platform, Monash University, Melbourne, Victoria, Australia.
authors.forename: Stuart K
authors.initials: SK
authors.lastname: Archer
authors.affiliation: Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.
authors.forename: Traude H
authors.initials: TH
authors.lastname: Beilharz
authors.affiliation: Monash Bioinformatics Platform, Monash University, Melbourne, Victoria, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Powell
authors.affiliation: EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.|Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.
authors.forename: Thomas
authors.identifier: 0000-0001-6273-784X
authors.initials: T
authors.lastname: Preiss
mesh_terms: D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D010443:Peptide Chain Termination, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D012333:RNA, Messenger