Vicent Pelechano1, Wu Wei2,3, Lars M Steinmetz1,2,3
1European Molecular Biology Laboratories, Genome Biology Unit, Germany;
2Stanford Genome Technology Center, Stanford University, USA;
3Department of Genetics, Stanford University School of Medicine
We have developed an optimized protocol (5PSeq) including unique molecular identifiers that allows measuring all 5’Phosphate mRNA degradation intermediates present in a sample. mRNA degradation intermediates have been previously analysed to identify RNA endonucleolitic cleavage sites for 5’-3’ RNA degradation mutants both in plants and yeast. However, what are the specific factors contributing to the presence of 5’P sites and how do their abundance vary across the genome and physiological conditions has not been studied.
We have investigated the 5’ positions of degradation mRNAs with 5PSeq in multiple physiological conditions and drug treatments in S. cerevisiae. Our genome-wide analysis shows that ribosomes act as a general barrier for mRNA degradation, suggesting that the previously proposed mRNA co-translation degradation is not only possible, but also pervasive in the genome. Analysis in other organism shows that this process is evolutionary conserved. By comparing 5PSeq with Ribosome Profiling, we show that the detailed study of the mRNA degradation intermediates produced by the endogenous RNA degradation machinery allows reconstructing ribosome dynamics in vivo. 5PSeq is a straightforward method that does not require any polyribosome fractionation or RNAse foot-printing and that can be easily applied to any previously purified RNA. Additionally this drug-free approach can be used to study ribosome pausing sites that in alternative methods such as Ribosomal Profiling are masked by the secondary effect of drugs like cycloheximide.
We believe that 5PSeq offers a new and complementary window to study global ribosome dynamics in conditions where the use of methods requiring the use of translation inhibition drugs or the isolation of polyribosomal fractions is not possible or advisable.
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