Fig 2: HnRNP H is a critical regulator of ACHE alternative splicing. (A) Immunoblotting (IB) with indicated antibodies of SH-SY5Y cell lysate after knockdown with siRNAs against control (si-Cont), HNRNPH (si-hnRNP H), HNRNPF (si-hnRNP F) and both HNRNPH and HNRNPF (si-hnRNP H/F). (B) RT-PCR of endogenous ACHE in SH-SY5Y cells treated with indicated siRNAs. (C) RT-PCR of ACHE minigene (MG) transfected in SH-SY5Y cells treated with si-hnRNP H/F with or without si-resistant hnRNP H cDNA. (D) Schematic of the ACHE minigenes (MG) carrying wild-type or mutant G-runs in exon 5a. G-runs are shown in bold and the introduced mutations are underlined. RT-PCR of wild-type and mutant constructs transfected in SH-SY5Y cell are shown. (E) Schematic of the ACHE minigene (MG) carrying the PP7 hairpin-loop substituting for two cis-acting G-runs in exon 5a (E5a-PP7-hp MG). HnRNP H is fused to the PP7 coat protein (inverted U shaped) to directly tether hnRNP H to the PP7 hairpin-loop. RT-PCR of minigenes cotransfected with the effector cDNAs in SH-SY5Y cells are shown. (B, C, D and E) Ratios of splicing isoforms of ACHE are shown in the stacked column graphs. Mean and SD are indicated (n = 3). P-values by two-way ANOVA are indicated above each graph. P-values by Tukey's multiple comparison test are indicated in Supplementary Table 2.

Fig 3: tRF-Gly-GCC binds to hnRNPF/H. (A) Biotin-oligo pull-downs from murine ES cell extracts. Silver-stained gel shows two replicates each for pull-downs using biotin-tRF-GG or biotin-tRF-Lys-CTT, as indicated. Arrow indicates ∼50-kDa band enriched in tRF-GG pull-downs. (B) Domain architecture of hnRNPF and hnRNPH1. (C) Mass spec peptide counts for hnRNPH in control, tRF-Lys-CTT, or tRF-GG pull-downs. (D) Western blots show hnRNPF/H recovery following tRF-GG or tRF-Lys-CTT pull-down. Pull-downs were washed four times for 3 min with 50 mM Tris (pH 8.0) supplemented with 100 mM (W1), 250 mM (W2), or 500 mM (W3) NaCl. (E) Enrichment of tRF-GG and two control RNAs in hnRNPF/H immunoprecipitates (and IgG controls) from mESCs, expressed as percent of input. Data show average and standard deviation from six data points: two biological replicates with three technical replicates each. (F) Gel shift analysis of hnRNPH1 binding to tRF-GG. A synthetic oligonucleotide corresponding to tRF-Gly-GCC (GCAJULGUGGUUCAGUGGDAGAAUUCUCGC) was labeled at the 3′ end using fluorescein 5-thiosemicarbazide, then incubated at 3 nM in equilibration buffer (0.01% Igepal, 0.01 mg/mL carrier tRNA, 50 mM Tris at pH 8.0, 100 mM NaCl, 2 mM DTT) for 3 h along with increasing concentrations of purified hnRNPH1 protein from 1.35 nM to 2000 nM. See also Supplemental Figure S7. (G,H) Fit of gel shift binding data for tRF-Lys-CTT and tRF-GG. Fitting the binding data yields an estimated Kd of hnRNPH1 of ∼220 nM for tRF-GG, and >1 μM for tRF-Lys-CTT. (I) Fluorescence polarization data for hnRNPH1 incubations with labeled tRF-GG. Polarization values against the protein concentrations are fit to the Hill equation using Igor Pro software.

Fig 4: HNRNPH1 interacts with G‐rich sequences in RBM3 poison exon in a temperature‐dependent manner. Related to Fig 5 Western blot and quantification of HNRNPH1 total protein levels (input HNRNPH1 normalised to GAPDH) and its abundance in spliceosomal protein SmB pulldown (normalised to spliceosomal protein SmD3) in HeLa cells at 37 and 32°C (72 h).Western blot and quantification of HNRNPH1 total protein levels (input HNRNPH1 normalised to ponceau measured total protein abundance) and its abundance in spliceosomal protein SmB pulldown (normalised to spliceosomal protein SmD3) in i‐neurons at 37 and 32°C (72 h).Representative images of HNRNPH1 staining of GFP‐RBM3 i‐neurons at 37°C or after 72 h cooling at 32°C. Graphs below the images show quantification of somal, nuclear and cytoplasmic intensity per unit area respectively. The decrease of HNRNPH1 signals at 32°C is due to the global attenuation of protein production, which is not seen after total protein or GAPDH normalisation in western blot in (B) and Fig 4A. N = 280 (37°C) and 262 (32°C) cells. Scale bar: 5 μm.Western blot of HNRNPH/F in HeLa cell input (total) lysate, IgG‐pulled down and HNRNPH1‐pulled down eluates at 37 and 32°C (48 h).Quantification of input‐normalised RBM3 mRNA levels in HeLa cell input (total), IgG‐pulled down and HNRNPH1‐pulled down RNA at 37 and 32°C (48 h).RNA G quadruplexes (rG4) within the RBM3 Exon 3–4 region are predicted using QGRS mapper. Deletion of the GGGG motif in the mutant RBM3 minigene is predicted to disrupt the rG4 structure overlapping this region.RT‐PCR of WT and delGGGG RBM3 minigenes in control or HNRNPH1‐overexpressing (OX) HEK293T cells treated with SMG1 inhibitor at 37°C. PSI values of RBM3 PE are shown in the graphs on the right. Data information: N = 3 biological replicates, except (B), which has N = 2. Mean ± SEM; n.s. (not significant), *(P < 0.05), **(P < 0.01), ***(P < 0.001); paired t‐test in (A), (B), (E), (G), unpaired t‐tests in (C). Source data are available online for this figure.

Fig 5: HNRNPH1 enhances RBM3 expression and RBM3 mRNA poison exon skipping on cooling. Related to Fig 4 Western blots and quantification of HNRNPH1 normalised to GAPDH in Control and HNRNPH1 KD Cas9WT i-neurons.Sashimi plots of the region between Exon 3 and 4 of RBM3 transcripts in control and HNRNPH1 knocked-down K562 and HepG2 cells, showing major alternatively spliced isoforms. Data are from ENCODE Project. 2 isogenic replicates are included.PSI values of RBM3 Exon 3a-S in control and HNRNPH1-knocked down K562 and HepG2 cells. RNA-Seq data from ENCODE Project, 2 isogenic replicates are included.qRT-PCR of HNRNPH1 mRNA normalised to GAPDH upon HNRNPH1 KD in HeLa cells.Western blots of FLAG-HNRNPH1 and GAPDH (loading control) in control and FLAG-HNRNPH1-expressed HEK293T cells at 37°C.Western blot of WT i-neurons transduced with lentivirus expressing BFP (control) or HNRNPH1-T2A-BFP (OX) at 37 and 32°C. The larger molecular weight of overexpressed HNRNPH1 is due to the additional amino acids between the C-terminus of HNRNPH1 and the T2A cleavage site.qRT-PCR of HNRNPH1 mRNA normalised to 18 s rRNA of WT i-neurons transduced with lentivirus expressing BFP (control) or HNRNPH1-T2A-BFP (OX) at 37 and 32°C.qRT-PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA (mean value of RBM3 exon 3 and exon 4–5) in SMG1 inhibitor-treated control and HNRNPH1-overexpressing (OX) WT i-neurons at 37 and 32°C.Median GFP intensity of control and HNRNPH1-overexpressing (OX) GFP-RBM3 i-neurons measured by flow cytometry.Representative dot plots of flow cytometry data showing BFP or HNRNPH1-T2A-BFP expression (X-axis) and GFP intensity of successfully transduced (BFP-positive) GFP-RBM3 i-neurons. Each data point represents one cell. Cells with high levels of BFP expression (top 5% in each well) are coloured in red (37°C) or blue (32°C), and the rest of BFP-positive cells are in grey. Y-axes are in the same scale and X-axes are scaled to the sample. Data information: N = 3 biological replicates. Mean ± SEM; n.s. (not significant), *(P < 0.05), **(P < 0.01), ***(P < 0.001); unpaired t-tests in (A), (G), (H), (I), FDR calculated by rMATS program in (C), paired t-test in (D). Source data are available online for this figure.