Transcriptome-wide measurement of translation by ribosome profiling

Abstract

Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose amino acid sequence corresponds to the codon sequence of an mRNA’s ORF. Translation is performed by the ribosome; therefore, in order to understand translation and its regulation we must be able to determine the numbers and locations of ribosomes on mRNAs in vivo. Furthermore, we must be able to examine their redistribution in different physiological contexts and in response to experimental manipulations. The ribosome profiling method provides us with an opportunity to learn these locations, by sequencing a cDNA library derived from the short fragments of mRNA covered by the ribosome. Since its original description, the ribosome profiling method has undergone continuing development; in this article we describe the method’s current state. Important improvements include: the incorporation of sample barcodes to enable library multiplexing, the incorporation of unique molecular identifiers to enable to removal of duplicated sequences, and the replacement of a gel-purification step with the enzymatic degradation of unligated linker.

Codon bias confers stability to humanmRNAs

“…It has been proposed that slower ribosome elongation rate modulated by low codon optimality affects the stability of mRNAs in yeast [14]. We then compared these scores with corresponding ribosome occupancies derived from ribosome profiling [34]. From the PCA, PC1 factor loadings of the codons were indicative of how much a particular codon contributed to the AT3-GC3 grouping, i.e., instability-stability ( Fig EV2A).…”

“…Ribosome profiling was performed according to the method previously described with following modifications [34]. RNA concentration of naïve HEK293T lysate was measured by Qubit RNA BR Assay Kit (Thermo Fisher Scientific).…”

“…Reads were aligned to human hg38 genome as described [34,88]. Reads were aligned to human hg38 genome as described [34,88].…”

Versatile Synthetic Route to Cycloheximide and Analogues That Potently Inhibit Translation Elongation

“…It is noteworthy that asingle treatment with 28 at 100 mm prior to lysis was sufficient to preserve the integrity of polysome.C urrently,t ypical polysome profiling experiments require ah igh concentration of 1 in all buffers after cell lysis because of the reversible binding of 1. [27] To understand how irreversible inhibition by 28 might be mediated, we conducted immunoprecipitation (IP) mass spectrometry (MS) experiments on RPL36a. Mass shifts corresponding to acylation of Lys22 by 28 were observed in both trypsin and chymotrypsin digests ( Figure 4E;s ee Figure S6).…”

RibORF: Identifying Genome‐Wide Translated Open Reading Frames Using Ribosome Profiling

“…The RNAs associated with protein complexes were purified for next-generation sequencing. The detailed experimental procedure was described in (McGlincy and Ingolia, 2017). However, during ribosome profiling procedure, no ribosome antibody was used to select ribosome-RNA complexes.…”