Fig. 2: IRE1α-specific cleavage of tRNAGly(GCC) in vitro.
From: 5′-tRNAGly(GCC) halves generated by IRE1α are linked to the ER stress response
a Northern blot analysis of in vitro cleaved tRNAGly(GCC) and tRNALys(CTT) by recombinant IRE1α (5 nM). Red arrow: prominent cleaved products of the tRNAGly(GCC) generated by IRE1α. The northern blot membranes were then stripped and reprobed with a 32P-5′-end-labelled probe specific for the tRNALys(CTT). Percentage of 5′-tRF compared to full-length tRNA are shown. Values are presented as the mean ± S.E.M (n = 3). P-values were obtained by one-way ANOVA with Dunnett’s multiple comparisons test. b Primer extension assay on tRNAGly(GCC) cleavage products in the presence of IRE1α in vitro. RE1α cleavage sites in the tRNAGly(GCC) are denoted by different letters (a–g). c In vitro cleavage of tRNAGly(GCC) by IRE1α. Purified tRNAGly(GCC) (20 ng) was incubated with recombinant IRE1α (5 nM) at 37 °C for 0.5 or 2 h. Secondary structure of mature tRNAGly(GCC) and IRE1α cleavage sites (a–g from Figs. 2b and 1–7 from Fig. 2c). Black arrows: position of the tRNAGly(GCC) cleavage site generated by IRE1α. Red arrow: major cleavage site by IRE1α. Source data are provided as a Source Data file.
