Fig. 7: Functional relevance of the RBM39–RNA interfaces in splicing.
From: Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39
a Schematic representation of the RBM39 isoforms used to rescue RBM39 KD. mRRM1.1 is mutated on RRM1 β-sheet surface, mRRM1.2 is mutated on the loop β2-β3, mRRM2 is mutated on the RRM2 RNA-binding interface and mRRM3 is mutated on its U2 snRNP binding interface. b Western blot analysis of RBM39 levels upon control knockdown (Ctrl KD), RBM39 knockdown (RBM39 KD), and rescue with FLAG-RBM39. 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 or 6 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 retained introns identified by RNA-Seq (MBD1, PAOLA and TPP1). Average values and standard deviations of three (mRRM1.2, mRRM2&3) or six (Ctrl KD, RBM39 KD, WT, mRRM1.1, mRRM2, mRRM3) biological replicates (n = 3 or 6) are shown. P values were computed from log-transformed ratios using two-sided unequal variances Welch’s t test65. e Agarose gel showing the efficiency of poison exon inclusion as assessed by RT-PCR. f 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.
