Widespread Co-translational RNA Decay Reveals Ribosome Dynamics

Abstract

Summary It is generally assumed that mRNAs undergoing translation are protected from decay. Here we show that mRNAs are in fact co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5’-3’ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5’phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress, which are dependent on the RNase Rny1. Our approach is simple and straightforward, and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns.

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

“…However, a recent genome-wide survey of monophosphorylated 59 RNA ends in yeast showed that XRN1 is also involved in the accumulation of 59-truncated RNA ends protected by ribosomes during RNA decay (Pelechano et al, 2015). Using the plant degradome data, we identified an array of 59-truncated RNA ends separated at 30-nucleotide intervals in uORFs and CDSs, implying an array of stacked ribosomes (Figures 2 and 4 to 6).…”

“…Pelechano et al (2015) also demonstrated that yeast 5Pseq data contain in vivo ribosome footprints, the products of cotranslation mRNA decay. The change in these ribosomeprotected termini captured by 5Pseq could reflect ribosome dynamics without the problems caused by translational inhibitors sometimes used in the generation of in vitro ribosome footprints.…”

“…High-throughput approaches for genome-wide profiling of RNA degradation intermediates that possess a free monophosphate at the 59 terminus have been developed by several groups and are variously named parallel analysis of RNA ends (PARE) (German et al, 2008), degradome sequencing (Addo-Quaye et al, 2008), genome-wide mapping of uncapped transcripts (Gregory et al, 2008), and 59 P sequencing (5Pseq) (Pelechano et al, 2015). Because intact mRNAs generally possess a 59 cap that blocks their ligation to RNA adaptors, truncated 59 RNA ends with a free monophosphate can be selectively sequenced by directly ligating poly(A) RNA with RNA adaptors.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Hence diverse cellular stress conditions can rapidly perturb global and mRNAspecific protein synthesis via multiple mechanisms to achieve stress-specific translational reprogramming. In addition to revealing translation changes under stress conditions, ribosomal profiling techniques have provided mechanistic insights into elongation through the use of distinct inhibitors (Lareau et al 2014) as well as by identifying specific pauses during elongation (Pelechano et al 2015).…”

“…Moreover, the mechanisms of translational reprogramming are stress specific. For example, the global response to amino acid starvation is dependent on Gcn2 and eIF2a phosphorylation, while the response to hydrogen peroxide involves inhibiting both initiation via Gcn2 and translation elongation (Shenton et al 2006), causing pausing at aspartic acid and serine codons according to ribo-seq experiments (Pelechano et al 2015); however, antioxidant-response mRNAs become well translated (Shenton et al 2006;Kershaw et al 2015). The response to glucose depletion is rapid and does not require eIF2a phosphorylation, with almost all ribosomes being lost from mRNAs within 1-2 min (Ashe et al 2000), perhaps via alterations in eIF4A function (Castelli et al 2011).…”