Fig. 2: G4 stabilizers suppress the inclusion of cold-repressed exons.

a Correlation of 5′-splice site G4 scores with delta PSI values after cold shock (HEK293T (35 °C–39 °C) and Hela (32 °C–37 °C)) or heat shock (Hela (40 °C–37 °C)). For each base surrounding the splice site, the G4 score is calculated using a 25 bp sliding window and then correlated with PSI. b Intersection of CIEs, NTs, and CREs comparing HEK293T and Hela cells (left panel), and proportions of the shared CREs containing sequences with different G4 scores (right panel). SS3/5 shows the maximum G4 scores for either the SS3 or the SS5 for each exon. CIE cold-induced exon, NT non-temperature sensitive exon, CRE cold-repressed exon. c Schematic depicting the position of CREs (red) in CDK4, IQSEC1, and FKBP15. Arrows indicate RT-PCR primers. It was generated with BioRender. d, e CRE inclusion level of CDK4 (d) and IQSEC1 (e) treated with DMSO or PDS at different temperatures in Hela (right) and HEK293T (left) cells. A representative gel image is shown. PCR products and sizes are indicated on the right. See Supplementary Fig. 2b, c (CDK4) and Supplementary Fig. 2e, f (IQSEC1) for quantifications. f PSI of RBM3 exon 3a in HEK293T and Hela RNA-Seq datasets from different temperatures. (see Fig. 1 and bioinformatics “Method”). Data represent n = 3 (HEK293T) and n = 4 (HeLa) independent biological replicates, with each data point corresponding to an individual replicate sequencing dataset. Statistical significance was determined using an unpaired two-tailed t-test. HEK293T: p = 0.0028 (35 °C vs. 39 °C); Hela: p = 0.0011 (32 °C vs. 37 °C), p < 0.0001 (37 °C vs. 40 °C). g Schematic of the RBM3 minigene designed to prevent translation to allow analysis of exon inclusion independent of NMD. PTCs: premature termination codons. This was generated with BioRender. h, i Exon 3a inclusion in the hRBM3 (h) and mRBM3 minigene (i) after G4 stabilizer PDS or control treatment in HEK293T cells. Upper gels depict representative minigene-specific RT-PCR results, and the lower part shows quantified results ((h) hRBM3: n = 3 for 33 °C, n = 9 for 35 °C (Ctr), n = 7 for 37 °C (Ctr) and n = 6 for PDS (35 °C and 37 °C) ; (i) mRBM3: n = 6 for 35 °C and 40 °C, n = 5 for 33 °C and 37 °C). h (hRBM3): p = 0.0007 (33 °C), p < 0.0001 (35 °C), p < 0.0001 (37 °C); i (mRBM3): p = 0.0005 (33 °C), p < 0.0001 (35 °C), p = 0.0014 (37 °C), p = 0.0004 (40 °C). j, k Sequence alignment of R1 and R3 across multiple mammals illustrated by a derived consensus sequence, the aligned sequences, and a sequence logo representation (see bioinformatics method). l Schematic illustrating the position of the G-rich elements R1 and R3 in the mRBM3 minigene and the sequence of the R13-2G mutant. It was generated with BioRender. m, n Splicing level of RBM3 exon 3a in WT and R13-2G mutant at different temperatures in both HEK293T (m) and N2a cells (n). Upper gels depict representative RT-PCR results, and the lower portion shows quantified results. In HEK293T (m), n = 5 (R13-2G at 39 °C) and n = 3 (others); in N2a cells (n), n = 4 (33 °C and WT at 37 °C) and n = 3 (others). m (HEK293T): p = 0.0362 (33 °C), p < 0.0001 (39 °C); n (N2a): p = 0.0008 (33 °C), p = 0.0121 (37 °C), p = 0.0063 (39 °C). In this figure, individual data points are shown with mean +/− SD. Statistical analysis was performed using unpaired two-tailed t-test. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, ***p ≤ 0.001 and **** denotes p ≤ 0.0001. Source data are provided as a Source Data file.