(A) A serial dilution (5-fold) yeast growth assay on rich media (YPD) and tunicamycin-containing media (+Tm) compares the growth of xrn1∆, trl1∆ (10x tRNA), and trl1∆ (RtcB) cells to resist protein-folding stress. Growth of wild-type cells is modestly affected by tunicamycin, whereas growth of xrn1∆ cells is partially inhibited by tunicamycin. Cells that lack ligase (trl1∆), and cells expressing E. coli RtcB RNA ligase in lieu of TRL1 (Tanaka et al., 2011) both fail to grow on media containing tunicamycin, and this growth defect is not affected by xrn1∆. (B) Expression of a UPR-responsive gene is compromised in RNA repair and decay mutants. RT-qPCR of mRNA for KAR2 (BiP), a direct target of Hac1 (Kohno et al., 1993), performed on total RNA from the indicated genotypes shows that wild-type cells induce KAR2 expression by 16-fold upon tunicamycin treatment. (Error bars are 95% confidence interval, n = 3; comparison bars represent p<0.01, Student’s t-test.) trl1∆ cells show an insignificant increase in UPR induction, whereas tpt1∆ cells have elevated KAR2 levels in the absence of tunicamycin, which does not change significantly after tunicamycin treatment. trl1∆ (RtcB) and xrn1∆ cells have a modest increase in expression, but not to the same degree as wild-type (p<0.01). (C) Excised HAC1 intron is stabilized in xrn1∆ and trl1∆ cells. Northern blot analysis using a probe to HAC1 intron reveals that excised intron (252 nt) and partially-degraded intron intermediates accumulate in xrn1∆ cells. Ligase-delete cells (trl1∆) also accumulate intron as a uniformly sized 252 nt product. In xrn1∆ and trl1∆ cells, intron accumulates in the absence tunicamycin. (D) Catalytic activity of Trl1 5′-kinase is required for 5′→3′ decay of excised HAC1 intron. Northern blot analysis using a probe to HAC1 intron shows that a missense mutation in the 5′-kinase domain of Trl1 (trl1-D425N) phenocopies the HAC1 accumulation of trl1∆ cells (lanes 4 and 6, also C). (E) The distributive 5′→3′ exonuclease Dxo1 can partially degrade HAC1 intron. Northern blot analysis for HAC1 intron on total RNA from dxo1∆ and dxo1∆ xrn1∆ cells shows that Dxo1 can partially degrade HAC1 intron when it accumulates in xrn1∆ cells (compare lanes 4, 6 and 8). (F) A slow-migrating intron species accumulates in trl1∆ cells expressing RtcB. Northern blot analysis of RNA from wild-type and trl1∆ cells shows that wild-type cells accumulate linear, partially degraded intron (lanes 1–4), whereas trl1∆ (RtcB), and trl1∆ xrn1∆ (RtcB) cells accumulate a slower-migrating species (~500 nt; lanes 5–8). (G) Cells expressing RtcB accumulate circular HAC1 intron. To test whether the slower-migrating band was circularized, total RNA was treated with RNase R and analyzed by northern blot. The slower-migrating species is largely protected from degradation, indicating it is a circle. Linear HAC1 intron and SCR1 (bottom) are degraded upon RNase R treatment (lanes 2, 4, 6 and 8). A panel of enhanced contrast shows that the slower migrating species in wild-type cells are circular, excised HAC1 introns, resistant to RNase R. Circular intron only occurs in samples from cells expressing a ligase. (H) The cytoplasmic exosome degrades HAC1 intron when 5′→3′ decay is disabled. Northern blot analysis using a HAC1 intron probe on total RNA from ski2∆ (a component of the cytoplasmic exosome) cells showed that ski2∆ cells accumulate excised HAC1 intron (lane 4) at levels similar to wild-type (lane 3). In trl1∆ ski2∆ cells, a lack of both kinase-mediated decay (trl1∆) and 3′→5′ decay (ski2∆) causes accumulation of excised intron relative to trl1∆ cells (compare lanes 6 and 8). (I) Catalytic activity of Trl1 ligase domain contributes to processing of excised HAC1 intron. We expressed a ligase-inactive Trl1 allele, trl1-K114A, as in Figure 2C. Expression of trl1-K114A (lanes 5 and 6) lead to a modest accumulation of intron, though not to the same extent as in the kinase-inactivated mutant (trl1-D425N) (lanes 3 and 4). The double missense mutant, trl1-K114A-D425N (lanes 7 and 8), exhibits intron accumulation similar that of trl1-D425N.