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, researchers from the University of Colorado School of Medicine 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. They 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). The researchers 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.
Schematic of 5′-hydroxyl (5′-OH) cloning. RNA cleavage generates two fragments with 2′,3′-cyclic phosphate and 5′-OH termini.