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.
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This work was supported by an ARC Discovery Grant (DP1300101928) and an NHMRC Senior Research Fellowship (514904) awarded to T.P. N.E.S. was supported by a Go8 European Fellowship. We acknowledge technical support from the Australian Cancer Research Foundation Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University) and S. Androulakis at the Monash Bioinformatics Platform.
The authors declare no competing financial interests.
Integrated supplementary information
Overview of the library construction steps used in TCP-seq, starting from the de-blocked RNA fragments isolated from the SSU, complete ribosome or total translated RNA fractions (Step 73), and ending with the amplified, size-selected strand-specific library (Step 128).
Representative alignments of reads (top sequences) to a reference (bottom sequences) are given and the nucleotides or gaps counted towards the footprint lengths is indicated in red. To the right of each alignment is its corresponding CIGAR representation, output by the aligner. 3′ poly(A) tracts that were mismatched with the reference (but not trimmed away in earlier steps due to an erroneous internal G or T base-call) were trimmed in this step also.
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Shirokikh, N., Archer, S., Beilharz, T. et al. Translation complex profile sequencing to study the in vivo dynamics of mRNA–ribosome interactions during translation initiation, elongation and termination. Nat Protoc 12, 697–731 (2017). https://doi.org/10.1038/nprot.2016.189
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