Genome-wide quantification of 5′-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics

• Published in: Nature protocols Vol. 11; no. 2; pp. 359 - 376
• Main Authors: Pelechano, Vicent, Wei, Wu, Steinmetz, Lars M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2016; Nature Publishing Group
• Subjects: 631/1647/514/1949; 631/337/1645/2020; 631/337/574; Analytical Chemistry; Biological Techniques; Codons; Computational Biology/Bioinformatics; Degradation; Degradation products; Depletion; Eukaryotic Cells - metabolism; Footprinting; Gene sequencing; Genetic research; Genetic translation; Genomes; Intermediates; Life Sciences; Messenger RNA; Methods; Microarrays; mRNA turnover; Next-generation sequencing; Oligonucleotides; Organic Chemistry; Properties; Protein Biosynthesis; protocol; Reverse transcription; Ribosomes; Ribosomes - metabolism; RNA sequencing; RNA Stability; rRNA; Saccharomyces cerevisiae; Translation
• DOI: 10.1038/nprot.2016.026
• ISSN: 1754-2189; 1750-2799

5PSeq is a method for studying ribosome dynamics based on co-translational mRNA decay. Genome-wide sequencing and quantification of 5′ phosphorylated mRNA degradation products allows the positions of the last translating ribosomes to be determined. Co-translational mRNA degradation is a widespread process in which 5′–3′ exonucleolytic degradation follows the last translating ribosome, thus producing an in vivo ribosomal footprint that delimits the 5′ position of the mRNA molecule within the ribosome. To study this degradation process and ribosome dynamics, we developed 5PSeq, which is a method that profiles the genome-wide abundance of mRNA degradation intermediates by virtue of their 5′-phosphorylated (5′P) ends. The approach involves targeted ligation of an oligonucleotide to the 5′P end of mRNA degradation intermediates, followed by depletion of rRNA molecules, reverse transcription of 5′P mRNAs and Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, and it is straightforward and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. Three days are required to generate 5PSeq libraries.