Genetic rescue of RtcB-mediated RNA ligation by yeast Trl1.
A, schematic representation of yeast (5′–3′ ligation) and mammalian (3′–5′ ligation) RNA ligation pathways and mammalian proteins involved in a putative 5′–3′ pathway. Yeast proteins are denoted in black, and mammalian proteins are underlined. B, images of methylene blue staining show that RtcB cKO ES cells survived in the absence of RtcB when yeast Trl1 was expressed. Western blotting confirmed that RtcB was depleted in E8H2 RtcB cKO ES cells after 4-OHT treatment and the rescued cells expressed Trl1. C, RT-qPCR analysis showed that the level of spliced tRNATyr dropped significantly in RtcB-depleted cells, whereas Trl1-expressing RtcB cKO cells exhibited a significantly higher level of tRNATyr. Expression levels were normalized to GAPDH mRNA levels and to the untreated control E8H2 cells. The error bars represent S.E. The results were subjected to two-tailed t tests. **, p < 0.01; ***, p < 0.001. D, FLAG-RtcB serves as a positive control for a complete rescue of XBP1s expression in E8H2. In RtcB Western blotting, the top bands are FLAG-RtcB, and the bottom bands are endogenous RtcB. ER stress was induced with thapsigargin (Tg) for 3 h. E, Western blots show that yeast Trl1 protein partially rescued the expression of XBP1s protein.