Fig. 4: PCBP1 promotes ESS of Carm1 which is inhibited by LincGET-guided paraspeckles.
a, Protein-binding site analysis of splicing acceptor and donor flanking exons 2 to 7 of Carm1 pre-mRNA. The sequences for E3-Mut and E3-C26 are shown. The known binding motifs are shown in the table (bottom left). b, RNA electrophoretic mobility shift assay analysis. Three biological replicates were performed. c, Combination charts showing changes in ESS of Carm1 pre-mRNAs. Data are mean and s.e.m. One-tailed Student’s t-tests were used for statistical analysis (n = 3 biological replicates). d, Immunofluorescence combined with RNA-FISH assays in L2C. Scale bar, 10 μm. Bottom left, relative fluorescence intensity. Right, numbers of LincGET speckles and PCBP1 speckles calculated by Imaris. Two-tailed Student’s t-tests were used for statistical analysis. e, Assays of qPCR following sequential co-IP. Data are mean and s.e.m. One-tailed Student’s t-tests were used for statistical analysis and P values are shown in Extended Data Fig. 7f (three biological replicates). N_P, sequential co-IP by anti-NONO antibody and then anti-PCBP1 antibody; P_N, sequential co-IP by anti-PCBP1 antibody and anti-NONO antibody. f, Single nucleus of L2C injected with fluorescently labeled LincGET mutants, sgRNAs–dCas9 complex and PCBP1. White triangles indicate sgRNA signals. Scale bar, 10 μm. n = 18(48/5/59) means that 18 embryos were analyzed, 59 Carm1 gene loci were detected, and 48 and 5 of them were covered by LincGET speckles and PCBP1, respectively. Bottom, relative fluorescence intensity. Rp values in d and f were calculated by Fiji/ImageJ.
