Genetic bypass of essential RNA repair enzymes in budding yeast.

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 mRNA exons during activation of the unfolded protein response (UPR). The RNA ligase Trl1 joins 2',3'-cyclic phosphate and 5'-hydroxyl RNA fragments, creating a phosphodiester linkage with a 2'-phosphate at the junction. The 2'-phosphate is removed by the 2'-phosphotransferase Tpt1. We bypassed the essential functions of and in budding yeast by expressing "prespliced," intronless versions of the 10 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 Δ and Δ mutants accumulate tRNA and splicing intermediates indicative of RNA repair defects and are hypersensitive to drugs that inhibit translation. Failure to induce the unfolded protein response in Δ cells grown with tunicamycin is lethal owing to their inability to ligate after its cleavage by Ire1. In contrast, Δ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced mRNA. We optimized a PCR-based method to detect RNA 2'-phosphate modifications and show they are present on ligated mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.