Fig 2: Transcriptome-wide and gene-specific analysis of the regulatory effect of hnRNP L paralogs.a Chart showing the decrease in expression of the genes depicted based on RNA-seq analysis post siRNA treatment. b, c Venn diagrams showing the overlap of differentially expressed genes and AS events upon hnrnpl and hnrnpll depletion as compared to scramble siRA treated cells. d Pie chart showing the fractions of differentially AS events that occur in both hnrnpl and hnrnpll depletion. e, f Genome browser view showing retention of introns in tjp1 and bptf genes upon hnrnpl depletion. g–j RNA was isolated from 293 T cells 72 h post-transfection with scramble siRNA, or siRNA against hnrnpl or hnrnpll. Also, k–n RNA was isolated from sihnRNPL expressing 293 T cells rescued with vector control (VC), hnRNP L, or hnRNP LL constructs. Specific primers were designed to detect the indicated genes and exons and individual alternative splicing events were checked by RT-PCR (h, l) (source data are provided as a Source Data file, the experiment was repeated two times all yielding similar results), measured by quantitative PCR, and represented by the ratios of depicted exons (i, j, m, n). For each sample n = 3 independent biological samples were examined in the same sequencing run. Data are presented as mean values with a standard error of the mean. An unpaired t test (two-tailed) was performed. p-Value < 0.05 was considered significant. p-Values are depicted on the top of the respective graphs. GAPDH was used for normalization.

Fig 3: The crystal structure reveals the molecular basis of hnRNP L-SETD2 interaction.a Ribbon representations of hnRNP L RRM2 bound to the SETD22167–2192 peptide. hnRNP L is colored in purple and the bound SETD22167–2192 peptide is colored in orange. b The SETD22167–2192 peptide is represented as ribbons on the molecular face of hnRNP L RRM2. The sidechains of SETD2Leu2188 and SETD2 Leu2189 are shown as sticks. Red and blue colors denote negative and positive surface charges, respectively. The electrostatic potential surfaces were generated with PyMol at the contouring value of the potential from −52.3 to 52.3 kTe−1. c–f Closeup views of the interactions between hnRNP L and the SETD22167–2192 peptide. (Left) The van der Waals surface views of hnRNP L-SETD22167–2192. hnRNP L (purple) and SETD22167–2192 (orange) are shown as ribbons with selected sidechains as sticks. The van der Waals surface of the hnRNP L is depicted as a semitransparent skin. The SETD2 peptide is represented as a stick diagram (orange). (Right) Hydrogen bonds are shown as black dashed lines.

Fig 4: Mutating key residues in hnRNP L and SETD2 disrupts their in vitro and in vivo binding.a ITC fitting curves of hnRNP L RRM2 WT and mutants with SETD22167–2192 peptide are shown. b, d, e Western blotting and silver staining of affinity-purified complexes of Halo-SETD2C WT and its mutants. In (b), mCherry-HA-hnRNP L proteins were used as prey. * denotes non-specific band. The experiment was repeated four times all yielding similar results. Source data are provided as a Source Data file. c, f ITC fitting curves of hnRNP L RRM2 with SETD2 peptides. For the arithmetic mean of KD values of the three independent experiments and the thermodynamic parameters see Supplementary Table 1. All ITC binding curves are shown in Supplementary Table 2.

Fig 5: Purification of hnRNP L RRM2 reveals SETD2 as an interactor.a Cartoon illustrating the truncation of hnRNP L along with the known domains. RRM RNA-recognition motif, NLS nuclear localization signal. b Microscopy images showing localization of mCherry-hnRNP L 162–321. The scale bar is 10 µm. The experiment was repeated at least four times all yielding similar results. c, d Halo purification was performed from extracts of 293T cells expressing Halo-hnRNP L 162–321. Input and eluted samples were resolved on gel followed by silver staining or western blotting. The expected band for the target proteins are depicted by arrows. HE high exposure, LE low exposure. The experiment was repeated at least two times all yielding similar results. e IPA (Ingenuity Pathway Analysis) of proteins enriched in Halo-hnRNP L 162–321 purification. f, g Table showing the dNSAFs ×100 of the listed protein. dNSAF distributed normalized spectral abundance factor.