Fig. 3: The RS domain of RBM39 is essential for RBM39 function in splicing.
From: Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39
a Schematic representation of the FLAG-tagged RBM39 construct lacking the RS domain. b Western blot analysis of RBM39 levels upon control knockdown (Ctrl KD), RBM39 knockdown (RBM39 KD), and rescue with FLAG-RBM39 lacking the RS domain (ΔRS). HeLa cell extracts were subjected to SDS–PAGE and Western blotting with anti‐RBM39 and anti‐FLAG antibodies. GAPDH served as a loading control. n = 3. 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. d Plot showing the number of nuclear foci counts of FLAG-RBM39 and FLAG-RBM39 ΔRS. P values were computed using two-sided t test, n = 3. e 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 biological replicates (n = 3) are shown. P values were computed from log-transformed ratios using two-sided unequal variances Welch’s t test65. f Agarose gel showing the effect of RS domain deletion on the splicing of the poison exon of RBM39 mRNA as assessed by RT-PCR. Below the gel, schematic representation of both RBM39 mRNA isoforms. g Barplot showing the percentage 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.
