title: 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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA Program in Molecular Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA.
authors.forename: Sally E
authors.initials: SE
authors.lastname: Peach
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.
authors.forename: Kerri
authors.initials: K
authors.lastname: York
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA Program in Molecular Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA jay.hesselberth@gmail.com.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Hesselberth
mesh_terms: D000604:Amino Acyl-tRNA Synthetases; D004722:Endoribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D059014:High-Throughput Nucleotide Sequencing; D012313:RNA / Q000737:chemistry; D059367:RNA Cleavage; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D056811:Unfolded Protein Response
chemical_list: D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012313:RNA; D004722:Endoribonucleases; D000604:Amino Acyl-tRNA Synthetases; C561935:RtcB protein, E coli