Fig. 4: Confirmation of specific association of HNRNPM and HNRNPH2 with 5′-tRH-Gly^GCC by RIP-seq analysis.

a Experimental design scheme for RIP-seq analysis. A2058 cells were transfected with expression vectors encoding indicated proteins (myc-IRE1α, HA-HNRNPM, FLAG-HNRNPH2), and cell extracts were prepared and immunoprecipitated with anti-HA or anti-FLAG antibodies. Three independent experiments were performed. b The proportion of RIP-seq enriched RNA fragments annotated to the indicated RNAs (n = 3). c Percentage of enriched tRF with HNRNPH2 (upper) or HNRNPM (lower) (n = 3). The pie chart represents the proportion of tRFs in each of the total tRF read counts in RIP-seq data. Mapped read counts from RIP-seq of tRF-Gly^GCC (d) or tRF-Cys^GCA (e) associated with HNRNPH2 (red) and HNRNPH2 (blue) were normalised to the corresponding read counts from the input. Data are presented as the mean ± S.E.M (n = 3). f Quantification of 5′-tRH-Gly^GCC enriched with HNRNPM or HNRNPH2 proteins. Co-immunoprecipitated RNAs from the indicated antibodies in A2058 cells were analysed by TaqMan-based qRT-PCR using designed probes on the 33-mer 5’-tRH-Gly^GCC. Data are presented as the mean ± S.E.M (n = 3). P-values were obtained by One sample t-test. g Pull-down of HNRNPM and HNRNPH2 using 5′-biotinylated tRH-Gly^GCC. TG (0.1 µM)-treated (+) or -untreated (−) KGN cell lysates were combined with 5′-biotinylated tRH-Gly^GCC or 5′-biotin-oligo A8 RNA (control). After allowing the binding of protein and RNA, streptavidin-coated beads were used to pull down the RNA. After washing, RNA-bound IRE1α, HNRNPM, or HNRNPH2 proteins were visualised using western blot. Source data are provided as a Source Data file.