Fig. 3: ER stress induces 5′-tRHs cleavage by tRNAGly(GCC).
From: 5′-tRNAGly(GCC) halves generated by IRE1α are linked to the ER stress response
a Northern blot analysis of tRNAGly(GCC) fragments in control KGN (WT) or IRE1α knockout-KGN cells (IRE1α−/−) (n = 3). Cells were treated with 0.1% DMSO, TG (0.1 μM) or TM (1 μg/ml) for 6 h and harvested. Total RNA was isolated and probed with a probe specific for the tRNAGly(GCC). The northern blot membranes were then stripped and reprobed with a 32P-5′-end-labelled probe specific for the tRNALys(CTT). Quantification of 5′-tRH-GlyGCC level is presented in the bottom panel. b (Upper) 5′-end of tRNAGly(GCC) fragment detected in a (KGN WT) determined by primer extension analysis. (Lower) Secondary structure of mature tRNAGly(GCC) and IRE1α cleavage sites at anticodon stem loop. Red arrow: major IRE1α cleavage site. c Validation of 5′-tRH enrichment following treatment of KGN WT and IRE1α−/− cells with 0.1 μM of TG by TaqMan-based real-time PCR. Data are presented as the mean ± S.E.M (n = 3). Northern blot analysis of tRNAGly(GCC) fragments in HeLa (d), A2058 (f), and TPC-1 (h) cells (n = 3). Total RNA was isolated and probed with a probe specific for the tRNAGly(GCC). The northern blot membranes were then stripped and reprobed with a 32P-5′-end-labelled probe specific for the tRNALys(CTT). Quantification of 5′-tRH-GlyGCC level presented in the bottom panel of d. Validation of 5′-tRH enrichment following treatment of HeLa (e), A2058 (g), and TPC-1 (i) cells with DMSO, TG or TM by TaqMan-based real-time PCR (n = 9). TaqMan-based real-time PCR of tRNAGly(GCC) or tRNALys(CTT) fragments in IRE1α knockdown or ANG knockdown HeLa (j), A2058 (k), and TPC-1 (l) cells with TG (0.1 μM), respectively. In a, d, f, and h, the expression of IRE1α and β-actin (loading control) was analysed by western blotting (WB). Ribonucleolytic activity of IRE1α was confirmed XBP1 splicing assay using RT-PCR analysis of unspliced/spliced (u/s) XBP1. P-values were obtained by one-way ANOVA with Turkey’s multiple comparisons. Red arrow: tRHs cleaved by IRE1α. Source data are provided as a Source Data file.
