Analysis and kinase-mediated decay of RNAs with 5´-hydroxyl termini

Abstract

RNA cleavage by metal ion-independent endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5 ́-hydroxyl (5 ́-OH) and 2 ́,3 ́-cyclic phosphate termini. However, RNAs with 5 ́-OH termini (5 ́-OH RNAs) are not detected by conventional sequencing methods, and 5 ́-OH RNAs cannot be directly degraded by canonical 5 ́-phosphate-dependent 5 ́→3 ́ exoribonucleases. To identify new 5 ́-OH RNA fragments, I exploited the unique ligation mechanism of E. coli RtcB RNA ligase to develop 5OH-seq, a novel method to capture and sequence 5 ́-OH RNAs. I applied 5OH-seq to budding yeast and captured known 5 ́-OH fragments produced by tRNA Splicing Endonuclease (SEN) during processing of intron-containing pre-tRNAs and by ribonuclease Rny1 during stress-induced cleavage of tRNA anti-codon loops. I 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 mRNA fragments were produced upstream of codons for highly electrostatic peptides, suggesting that the fragments may be generated by co-translational mRNA decay, such as no-go decay (NGD). 5 ́-OH RNAs must be phosphorylated to become substrates for 5 ́-phosphate- dependent 5´→3 ́ exoribonucleases, a process I term “kinase-mediated decay” (KMD). During the unfolded protein response (UPR), the ER-localized Ire1 endoribonucleasecleaves HAC1 mRNA, yielding 2 ́,3 ́-cyclic phosphate and 5 ́-OH RNA fragments that are ligated by Trl1 tRNA ligase to produce mature HAC1 mRNA and activate downstream target genes. The HAC1 fragments cleaved by Ire1 have previously been identified with 5 ́-phosphate (5-PO4) termini in vivo in xrn1∆ yeast, suggesting the HAC1 fragments are KMD substrates. I found that HAC1 3 ́-exons accumulate with 5 ́-PO4 termini in xrn1∆ yeast when Trl1 is present, but not kinase-dead Trl1-D425N or RtcB ligase, suggesting that Trl1 phosphorylates the HAC1 3 ́-exon for decay by Xrn1. Failure to degrade the HAC1 3 ́-exon promotes aberrant splicing and UPR activation without stimulus. In summary, this thesis describes a method to directly capture and sequence 5 ́-OH RNAs (5OH-seq), a comprehensive analysis of 5 ́-OH RNAs in budding yeast, and evidence for a unified theory of 5 ́-OH RNA degradation by kinase-mediated decay (KMD). I also present preliminary data on the abundance of 5 ́-OH RNAs in mammalian tissues that lack the RNA 5 ́-kinase CLP1.