Fig. 2: Contributions of the three RBM39 RNA recognition motifs to splicing regulation.
From: Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39
a Western blot analysis of RBM39 levels upon control knockdown (Ctrl KD), RBM39 knockdown (RBM39 KD), FLAG-RBM39 rescue (WT rescue) or rescue performed with RBM39 lacking each one RRM (ΔRRM1, ΔRRM2 and ΔRRM3 rescues). HeLa cell extracts were subjected to SDS–PAGE and Western blotting with anti‐RBM39 and anti‐FLAG antibodies. Tubulin 1A2 served as a loading control. b Schematic representation of the FLAG-tagged RBM39 deletion constructs. c Immunofluorescence analysis of FLAG-RBM39 constructs upon transient expression in HeLa cells. Exogenous RBM39 was visualised using anti-FLAG antibodies and nuclei were counterstained with DAPI, n = 3. d RT-qPCR measurements displayed as the ratio of spliced to unspliced isoform for three intron retention events identified by RNA-Seq (MBD1, PAPOLA and TPP1). Average values and standard deviations of three (ΔRRM1, ΔRRM2, ΔRRM3) or four (Ctrl, RBM39 KD, WT) biological replicates (n = 3 or 4) are shown. P values were computed from log-transformed ratios using two-sided unequal variances Welch’s t test65. e Schematic representation of both RBM39 mRNA isoforms. In the longer isoform, the exon 2b is included and introduces a premature termination codon (PTC). f Agarose gel showing the effect of RRM deletion mutants on the splicing of the poison exon of RBM39 mRNA as assessed by RT-PCR. g Barplot showing the percent of exon 2b inclusion in the different conditions. Average values and standard deviations of three biological replicates (n = 3) are shown. P values were computed using two-sided t test.
