RNA repair enzymes catalyze rejoining of an RNA molecule after cleavage of phosphodiester linkages. RNA repair in budding yeast is catalyzed by two separate enzymes that process tRNA exons during their splicing and HAC1 mRNA exons during activation of the unfolded protein response. The RNA ligase Trl1 joins 2′,3′-cyclic phosphate and 5′-hydroxyl RNA fragments, creating a new phosphodiester linkage with a 2′-phosphate at the junction. The 2′-phosphate is subsequently removed by the 2′-phosphotransferase Tpt1, which catalyzes phosphate transfer to NAD+, producing nicotinamide and a unique ADP ribose metabolite. We bypassed the essential functions of TRL1 and TPT1 in budding yeast by expressing “pre-spliced,” intronless versions of the ten normally intron-containing tRNAs, indicating this repair pathway does not have additional essential functions. Consistent with previous studies, expression of intronless tRNAs failed to rescue the growth of cells with deletions in components of the SEN complex, implying an additional essential role for the splicing endonuclease. The trl1∆ and tpt1∆ mutants accumulate tRNA and HAC1 splicing intermediates indicative of specific RNA repair defects and are hypersensitive to drugs that inhibit translation. As expected, failure to induce the unfolded protein response in trl1∆ cells grown with tunicamycin is lethal owing to their inability to ligate HAC1 after its cleavage by Ire1. In contrast, tpt1∆ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced HAC1, suggesting that ligated but 2′-phosphorylated mRNA is decoded by the ribosome. Finally, we optimized a PCR-based method to detect RNA 2′-phosphate modifications and show that they are present on ligated HAC1 mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.

New tools for reproducible exploratory data analysis of large datasets are important to address the rising size and complexity of genomic data. We developed the valr R package to enable flexible and efficient genomic interval analysis. valr leverages new tools available in the ‘tidyverse’, including dplyr. Benchmarks of valr show it performs similar to BEDtools and can be used for interactive analyses and incorporated into existing analysis pipelines.

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.

The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes.