Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

Figures

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  Figure 1. Dichromatic fluorescence alternative splicing reporter for Ttn to monitor the RBM20 activity in splicing regulation. (A) Schematic representation of the Ttn reporter minigene TtnE50-E51E218-E219-EGFP/mCherry (top) and mRNAs derived from it (bottom). Two genomic fragments Ttn E50-E51 and E218-E219 were inserted between human histone H2B cDNA and the EGFP/mCherry cassette. Expression of E51E218-EGFP and ΔE51E218-mCherry indicates inclusion and skipping of a chimeric exon E51E218, respectively. (B) Microphotographs of HeLa cells co-transfected with the fluorescence Ttn splicing reporter minigene and an empty vector or an expression vector for the wild-type (WT) or mutant RBM20 protein. Fluorescence of EGFP and mCherry is pseudo-colored in green and magenta, respectively. Scale bar, 100 µm. (C) RT-PCR analysis of the Ttn splicing reporter minigene co-expressed with an empty vector or an expression vector for the wild-type (WT) or mutant RBM20 protein in HeLa cells. Representative gel-like presentation (left) and calculated inclusion levels (right) are indicated. Error bars indicate standard errors of the means. #p < 0.001 and **p < 0.01 to WT (n = 3 biological replicates, one-way…

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  Figure 2. The RRM domain and the zinc finger domains of RBM20 are not critical for splicing regulation of the Ttn reporter. (A) Domain structure of mouse RBM20 protein. Names and positions of the domains are indicated. These domains are deleted in the mutants used in panels (B–D). (B) Microphotographs of HeLa cells co-transfected with the fluorescence Ttn splicing reporter minigene and an expression vector for the wild-type (WT) or deletion mutant RBM20 protein. The images are presented as in Fig. 1B. (C,D) RT-PCR analysis of the Ttn splicing reporter minigene co-expressed with an expression vector for the wild-type (WT) or mutant RBM20 protein in HeLa cells. The data are presented as in Fig. 1C.

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  Figure 3. The RSRSP stretch is critical for nuclear localization of and not splicing regulation by RBM20. (A) Amino acid sequence alignment of the RS-rich region of RBM20 proteins from human (Accession No. NP_001127835), mouse (NP_001164318), rat (NP_001101081), chicken (XP_015144459) and frog (XP_002942947) by Clustal W and manual adjustment. Amino acid residues that match the human RBM20 residues are shaded. The RSRSP stretch is boxed. Asterisks indicate evolutionarily conserved arginine (R), serine (S) and proline (P) residues. A line above the alignment indicates the sequence of a phospho-peptide used for raising anti-phospho-RBM20 antibody. (B) Immunofluorescence staining of FLAG-tagged wild-type RBM20 and substitution mutants RBM20S637A, RBM20S639A and RBM20S637A/S639A ectopically expressed in HeLa cells. DAPI staining of the same field is indicated below each panel. Scale bar, 20 µm. (C) Immunofluorescence staining of FLAG-tagged wild-type RBM20, RBM20S637A/S639A and RBM20S637A/S639A with three tandem copies of SV40 NLSs (NLS-RBM20S637A/S639A) in HeLa cells. The images are shown as in (B). (D) RT-PCR analysis of the Ttn splicing reporter minigene co-expressed with an expression…

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  Figure 4. RBM20 is constitutively phosphorylated on the RSRSP stretch and some other site(s). (A) Pro-Q Diamond staining (left) and negative staining (right) of immuno-precipitated and polyacrylamide gel-electrophoresed FLAG-tagged RBM20WT and RBM20S637A/S639A expressed in HEK293T cells. The precipitated proteins were sequentially incubated with heat-inactivated (−) or active (+) calf intestine protein phosphatase (CiAP) and lambda protein phosphatase (λPP). (B) Western blot detection of immuno-precipitated FLAG-tagged wild-type RBM20(517–657), RBM20(517–657)S637A, RBM20(517–657)S639A and RBM20(517–657)S637A/S639A expressed in HEK293T cells. The proteins were separated by either standard SDS-PAGE (left) or SDS-PAGE with 25 µM Phos-tag (right) following incubation without (−) or with (+) λPP. (C) Western blot detection of immuno-precipitated FLAG-tagged full-length RBM20 and RBM20S637A/S639A expressed in HEK293T cells following incubation without (−) or with (+) λPP. Antibodies used are indicated on the right.

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  Figure 5. Rbm20S637A knock-in mice are defective in producing the N2B isoform of titin in the heart. (A) Sequence analysis of Rbm20 mRNAs from hearts of a wild-type mouse (Wt) and a heterozygote (Het) and a homozygote (Homo) of Rbm20S637A knock-ins. (B) Vertical SDS-agarose gel electrophoresis and CBB staining of cardiac proteins from the hearts of 7-week-old Rbm20S637A knock-in mice in one litter. Genotypes of the individual mice are indicated above. Titin isoforms (N2B, N2BA and N2BA-G) and myosin heavy chain (MHC) are indicated. (C) RT-PCR analysis of Ttn mRNAs in the mice shown in (B) with E50 forward, E51 reverse and E219 reverse primers (top), E50 forward, E215 forward and E219 reverse primers (middle) and E115 forward, E215 forward and E219 reverse primers (bottom). Splicing patterns of the PCR products and names of corresponding titin isoforms are indicated on the right. (D) RT-PCR analysis of Ldb3 (top) and Camk2d (bottom) mRNAs as shown in (C).

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  Figure 6. Evaluation of RBM20 mutations identified in DCM patients. (A) Point mutations in the coding region of the human RBM20 gene analyzed in this study. Domain structure of the human RBM20 protein and positions of the mutations are indicated. Numbers in parentheses indicate positions of corresponding residues in the mouse RBM20 protein. (B) RT-PCR analysis of the Ttn splicing reporter minigene co-expressed with an expression vector for the wild-type (WT) or mutant RBM20 protein in HeLa cells. The data are presented as in Fig. 1C. (C) Western blot analysis of FLAG-tagged wild-type and mutant RBM20 proteins ectopically expressed in HEK293T cells with anti-phospho-RBM20 (top) and anti-FLAG (bottom) antibodies. (D) Immunofluorescence staining of FLAG-tagged wild-type and mutant RBM20 proteins ectopically expressed in HeLa cells. DAPI staining of the same field is indicated below each panel. Scale bar, 20 µm.