Fig. 8: hnRNPM collaborates with BCAS2 to regulate alternative splicing.

a Schematic diagram of the hnRNPM immunoprecipitation-mass spectrometry (IP-MS) workflow for profiling hnRNPM-interacting proteins in GV oocytes. Schematic illustration of mice was adapted from SciDraw. The oocyte diagram is a modified version of an original Servier Medical Art resource. All modified images are used under a CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/). b GO enrichment analysis of the identified hnRNPM-interacting proteins by Metascape. c Protein-protein interaction (PPI) network of hnRNPM-interacting proteins involved in RNA splicing. d In vivo co-immunoprecipitation (Co-IP) assays in mouse oocytes to determine whether the interaction between hnRNPM and BCAS2 is RNA-dependent, using treatments with RNasin (ribonuclease inhibitor) or RNase A. All samples presented within this figure panel derive from the same experiment and were processed in parallel. e The localization of BCAS2 in the primordial, primary, secondary, and antral follicles. The lower panels show magnified views of the indicated regions (boxes above). Scale bars: 50 μm. f Venn diagrams showing the overlap of abnormal AS genes and events in Hnrnpm and Bcas2 cKO oocytes. The overlaps of 98 common genes and 57 identical splicing events were both highly significant by hypergeometric test (p = 2e-27 and p = 4e-27, respectively). g–i Sashimi plots showing the splicing patterns of Cenpf, Prc1, and Hdlbp in control, Hnrnpm cKO, and Bcas2 cKO oocytes. j PCR validation of splicing events in (g–i). Schematic showing the alternatively spliced exons. k Bar plots display the percent spliced- in (PSI) values, calculated as PSI = splice_in/(splice_in + splice_out). Two-sided Student’s t-tests. Data are presented as mean ± SEM. n = 3. For Bcas2 ctrl and cKO group, p = 0.003473 (Cenpf), 0.000519 (Prc1), 0.007735 (Hdlbp). For Hnrnpm ctrl and cKO group, p = 0.009008 (Cenpf), 0.012079 (Prc1), 0.001602 (Hdlbp). l, m RIP-PCR and qPCR analyses of hnRNPM and BCAS2-interacted transcripts in P21 ovaries. RNA immunoprecipitation followed using qPCR (RIP-qPCR) was performed to detect Cenpf, Prc1, and Hdlbp transcripts co-precipitated with anti-hnRNPM antibodies using anti-IgG as a negative control in P21 mouse ovaries. Two-sided Student’s t-tests. Data are presented as mean ± SEM. n = 5. n Western blotting confirmed the high knockdown efficiency of hnRNPM in HEK293T cells. o, p RIP-PCR and qPCR analyses revealed BCAS2-RNA interactions in control and HNRNPM knockdown 293T cells. The association of CENPF, PRC1, and HDLBP transcripts with BCAS2 was examined using RIP-PCR (o) and qPCR (o). Two-sided Student’s t-tests. Data are presented as mean ± SEM. n = 5. p = 0.000037 (PRC1). p < 0.000001 (CENPF, HDLBP). All samples presented within a single figure panel (d, j, n) derive from the same experiment and were processed in parallel. Representative results shown in (d, e, j, l, n, o) were obtained from at least three independent experiments with similar results. Source data (d, j–p) are provided as a Source data file.