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 known 5′-OH fragments produced by tRNA Splicing Endonuclease (SEN) during processing of intron-containing pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction of the unfolded protein response (UPR). We identified numerous novel 5′-OH fragments derived from mRNAs: some 5′-OH mRNA fragments were derived from single, localized cleavages, while others were likely produced by multiple, distributed cleavages. Many 5′-OH fragments derived from mRNAs were produced upstream of codons for highly electrostatic peptides, suggesting that the fragments may be generated by co-translational mRNA decay. Several 5′-OH RNA fragments accumulated during the induction of the UPR, some of which share a common sequence motif that may direct cleavage of these mRNAs. This method enables specific capture of 5′-OH termini and complements existing methods for identifying RNAs with 2′,3′-cyclic phosphate termini.

Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis

“…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 .…”

Translation of poly(A) tails leads to precise mRNA cleavage

“…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.…”

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

“…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).…”