Pelechano, V., Wilkening, S., Jarvelin, A.I., Tekkedil, M.M.iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Counting absolute numbers of molecules using unique molecular identifiers. The ribosome profiling strategy for monitoring translation in vivoby deep sequencing of ribosome-protected mRNA fragments. Ingolia, N.T., Brar, G.A., Rouskin, S., McGeachy, A.M.Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes-GMUCT 2.0. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes. Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae. A link between RNA metabolism and silencing affecting Arabidopsis development. Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Addo-Quaye, C., Eshoo, T.W., Bartel, D.P.An efficient method for genome-wide polyadenylation site mapping and RNA quantification. Extensive transcriptional heterogeneity revealed by isoform profiling. Genome-wide identification of transcript start and end sites by transcript isoform sequencing. Widespread co-translational RNA decay reveals ribosome dynamics. Co-translational mRNA decay in Saccharomyces cerevisiae. Hu, W., Sweet, T.J., Chamnongpol, S., Baker, K.E.RNA degradation in Saccharomyces cerevisae. The intimate relationships of mRNA decay and translation. Three days are required to generate 5PSeq libraries. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. 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. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. 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. 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. 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.
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