Fig. 1: Small RNA-seq analysis of IRE1α-induced tRFs in vivo.

a Volcano plot depicting differentially expressed 5’-tRFs in WT and IRE1α-overexpressing KGN cells (KGN-IRE1α^oe) (n = 3). tRNA gene annotation: ‘W-X:Y^Z’ (W: amino-acid; X: anticodon; Y: cleavage site; Z: unique gene identifier). Log₂ fold change >1.5 and p < 0.001 was used as cut-off for significance (yellow box). b Based on the small RNA-seq analysis in a, cleavage sites at the anticodon loop in the secondary human tRNA^Gly(GCC) and tRNA^Cys(GCA) structures (n = 3). Red: acceptor stem at 5′-end; Purple: D loop; light green: anticodon loop; dark green: anticodon; yellow: T loop; blue: CCA tail at 3′-end. Numbering in the anticodon indicates the 3′-end positions of the tRFs; percent indicates the proportion of 5’-tRFs in total 5’-tRFs (log₂ fold change >1.5 and p < 0.001). c Northern blot analysis of tRNA fragments in KGN cells following IRE1α overexpression KGN cells were transfected with plasmid encoding myc-tagged IRE1α for 24 h, total RNA was extracted for analysis of 5′-tRNA fragments by northern blotting. The expression of IRE1α and GAPDH (loading control) was analysed by western blotting. Ribonucleolytic activity of IRE1α was confirmed XBP1 splicing assay using RT-PCR analysis of unspliced/spliced (u/s) XBP1. Red arrow: 5′-tRFs from tRNA^Gly(GCC) generated by IRE1α. M: size marker. Percentage of 5′-tRF compared to full-length tRNA are shown. Data are presented as the mean ± S.E.M (n = 3). P-values were obtained by unpaired two-tailed t-test. d (Left) Primer extension analysis of 5′-end of tRNA^Gly(GCC) fragment in KGN cells. KGN cells were transfected with a plasmid encoding IRE1α or kinase defected mutant (IRE1α-K599A). (Right) Secondary structure of mature tRNA^Gly(GCC) and IRE1α cleavage sites at anticodon. Numbering in the anticodon indicates the positions of mature tRNA nucleotides. Red arrow: prominent cleaved products of the tRNA^Gly(GCC) generated by IRE1α. Source data are provided as a Source Data file.