High-throughput 5’P sequencing enables the study of degradation-associated ribosome stalls

Abstract

RNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is intimately connected to the translation process up to the degree that 5'-3' co-translational mRNA degradation can be used as a proxy for ribosome dynamics. Here we present an improved high-throughput 5'P RNA sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable and uses affordable duplex-specific nuclease based rRNA depletion. We investigate in vivo ribosome stalls in Saccharomyces cerevisiae and Schizosaccharomyces pombe focusing on translation termination. We show that degradation intermediates of mRNAs with high termination pausing often use TGA as stop codon preceded by Lys and have relatively shorter poly(A) tails. We investigate the regulation of this process after glucose deprivation and reveal that, in addition to translation initiation, glucose starvation also leads to ribosome stall at the stop codon. Finally, we investigate gene-specific termination pausing across multiple conditions and show that it is a highly regulated process that crosstalks with codon optimality and the length of poly(A) remaining after deadenylation. In summary, HT-5Pseq is an improved and cost-effective approach that allows to investigate the crosstalk between RNA degradation and the translation process and that is particularly useful to examine its coupling at the level of translation termination. Key points:• HT-5Pseq facilitates high throughput investigation of 5'P mRNA degradation intermediates.• Gene-specific ribosome pause at termination level is common and environmentally regulated.• Ribosome stall at termination level are associated with TGA usage and shorter deadenylated poly(A) tails.

Improved computational analysis of ribosome dynamics from 5’P degradome data using fivepeseq

“…This effect was not dependent on genotype (early flowering, flc-3, or late flowering, FLC) or the tissue under study (cotyledons from day 6, apices from days 9 and 11, and leaves from days 14, 23, 32 and 33) ( Figure 5A). Increased termination stall during A. thaliana development is in line with previously observed reduction in polysome associated mRNAs (45), and the increased ribosome termination stalls that we previously described in budding yeast during limited nutrient conditions or stationary phase (3,8). Interestingly, apices showed subtle preference for 5'P counts 17nt upstream from the stop codon, as opposed to the general -16 nt preference in the other samples ( Figure 5A, Figure S3E).…”

“…Although initially considered independent events, multiple evidence has now demonstrated that translation and mRNA decay are interconnected processes and that co-translational mRNA degradation is a general phenomenon (2)(3)(4)(5). The interaction between the translation and decay machinery occurs so close that the positions of 5'P co-translational mRNA degradation intermediates can be used as a proxy for ribosome dynamics, as we and others have shown in yeast (6)(7)(8) and plants (4,9,10). In particular, XRN1driven 5'-3' mRNA degradation is linked to the movement of the last translating ribosome allowing to obtain an in vivo footprint of the ribosome position.…”

“…However, investigating the 5'P degradome is a very useful complementary approach and allows to obtain a drug-free measurement of in vivo ribosome position (omitting in vitro RNA degradation) and to focus on those mRNAs that are undergoing co-translational decay (3,8,14). Over the years, multiple techniques have been developed to investigate 5'P mRNA degradation intermediates (6)(7)(8)(15)(16)(17)(18).…”

“…Similar approaches have also been used to investigate endonucleolytic cleavage in budding yeast. To investigate the link between the ribosome position and mRNA decay, we developed 5PSeq (6,7) and more recently, an improved version of this approach, HT-5PSeq (8). Interestingly, the reanalysis of 5'P data originally generated to investigate m i R N A m e d i a t e d e n d o n u c l e o l y t i c c l e a v a g e , demonstrated that general 5'P degradome sequencing informs about ribosome position also in plants (4,18,19).…”

“…Such preprocessing steps for read length selection and adjustment, although important for ribosome profiling, are not relevant for 5'P mRNA degradome datasets, as the latter usually produce longer reads (e.g. more than 50nt (8)), where only the 5' site is functionally relevant, and do not require 5' adjustment (6)(7)(8). Thus, applying ribosome profiling analysis tools to 5´P degradome sequencing data is complex and would require computational preprocessing to trim the reads to 28-32 nt, limiting the advantages of long-read sequencing and requiring bioinformatics expertise.…”