Journal
NUCLEIC ACIDS RESEARCH
Volume 43, Issue 17, Pages E108-U18Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv536
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Funding
- Damon Runyon-Rachleff Innovation award
- Research Scholar Grant from the American Cancer Society
- National Institutes of Health Training [T32 GM008730]
- University of Colorado Cancer Center Grant [P30 CA046934]
- Internal University funds
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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 bymultiple, 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.
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