2015
DOI: 10.1093/nar/gkv536
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Global analysis of RNA cleavage by 5′-hydroxyl RNA sequencing

Abstract: RNA cleavage by some endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5′-hydroxyl (5′-OH) and 2′,3′-cyclic phosphate termini. To identify 5′-OH RNA fragments produced by these cleavage events, we exploited the unique ligation mechanism of Escherichia coli RtcB RNA ligase to attach an oligonucleotide linker to RNAs with 5′-OH termini, followed by steps for library construction and analysis by massively parallel DNA sequencing. We applied the method to RNA from budding yeast and captured… Show more

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Cited by 42 publications
(42 citation statements)
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“…In this case, ribothrypsis products are immediately accessible to XRN1, which degrades RNAs bearing 5′-P, and ribothrypsin is likely a protein whose cleaved products bear 5′-P (3′-fragment) and 3′-OH (5′-fragment). However, we note that the Hesselberth lab was able to selectively capture and sequence, from budding yeast, the small amounts of RNAs bearing 5′-OH termini by employing RtcB RNA ligase and found that such fragments originated from CDS regions of numerous mRNAs 60 . Intriguingly, 5′-OH fragments seemed to accumulate upstream of codons for acidic and basic aminoacids 60 .…”
Section: Discussionmentioning
confidence: 94%
“…In this case, ribothrypsis products are immediately accessible to XRN1, which degrades RNAs bearing 5′-P, and ribothrypsin is likely a protein whose cleaved products bear 5′-P (3′-fragment) and 3′-OH (5′-fragment). However, we note that the Hesselberth lab was able to selectively capture and sequence, from budding yeast, the small amounts of RNAs bearing 5′-OH termini by employing RtcB RNA ligase and found that such fragments originated from CDS regions of numerous mRNAs 60 . Intriguingly, 5′-OH fragments seemed to accumulate upstream of codons for acidic and basic aminoacids 60 .…”
Section: Discussionmentioning
confidence: 94%
“…Both processes have been observed on ORF-internal polylysine reporters as well as reporters where the ribosome translates authentic poly(A) tail (Tsuboi et al 2012;Shao et al 2013;Shcherbik et al 2016). More generally, runs of polybasic residues in the genome have been reported to increase mRNA cleavage, possibly due to the NSD/NGD pathway (Peach et al 2015). However, it remains unclear whether these pathways operate independently or specialize for particular substrates.…”
Section: Introductionmentioning
confidence: 99%
“…PNKs target the 5 ′ -terminus of DNA and RNA substrates and catalyze the transfer of the γ-phosphate from nucleoside triphosphate (NTP) to the 5 ′ -hydroxyl terminus of the polynucleotide substrate. The 5 ′ -phosphorylation status of DNA and RNA is critically important in the cell, thus underscoring the need for the PNK family of enzymes (Peach et al 2015). For example, phosphorylation of 5 ′ -hydroxyl termini following DNA damage is required for subsequent DNA repair (Weinfeld et al 2011); whereas RNA turnover by the eukaryotic Xrn 5 ′ -exonuclease family requires a 5 ′ -phosphate for RNA degradation (Heindl and Martinez 2010;Jinek et al 2011).…”
Section: Introductionmentioning
confidence: 99%