Figure 1.
Phylogenetic analysis of RBM4 proteins.
A phylogenetic tree of various metazoan RBM4 orthologs was created with the Clustal X program based on alignment of their full-length amino acid sequences. Branch lengths are drawn to scale, and weights for each RBM4 homolog sequence are given; a value of 0.01 represents a difference of 1% between two sequences. Each colored bracket indicates a group of species, in which RBM4 orthologs/homologs contain recognizable domains (RRM or zinc-finger; blue), the potential phosphorylation site RD/ERSP (red), or low-complexity sequences (brown). Detailed information for the low-complexity sequences is shown in Figure 2.
Figure 2.
The C-terminal domain of RBM4 proteins.
The C-terminal ∼150 residues of RBM4 homologs are shown. Colored boxes are low-complexity motifs including Ala-rich (pink), Pro-rich (yellow), Ser-rich (olive green), AP/PA (blue), and AS/SA (grey). RS dipeptides are highlighted in red; putative phosphorylation sites (RD/ERSP) are underlined.
Figure 3.
Drosophila Lark acts as a splicing regulatory protein.
The expression vector of FLAG-tagged human RBM4a or Drosophila Lark was cotransfected with a splicing minigene reporter, adenovirus E1a (A), Pax6 (B) or Kif1b (C), into HeLa (A) or HEK293 (B and C) cells; the minigenes are shown in each panel. Mock represents the empty expression vector. To detect the splicing products, RT-PCR was performed using total RNA as template, and then the resulting products were separated on polyacrylamide gels (E1a and Pax6) or agarose gels (Kif1b). Except for Kif1b, RT-PCR products were further blotted onto membranes, and detected by hybridization with 32P-labeled specific probe (Table 1). For E1a, the bar graph shows the relative abundance (%) of the major splicing products. For Pax6 and Kif1b, relative exon inclusion efficiencies are indicated below the gel or blot. Exon inclusion efficiency was calculated as exon-included RNA/total RNA. Bar graph shows exon inclusion fold relative to the mock; bar graph shows average values with standard deviations obtained from three independent experiments. (D) Immunoblotting using anti-FLAG shows overexpressed FLAG-tagged Lark and RBM4a.
Figure 4.
Evaluation of the alternative splicing activity of RBM4 homologs.
In vivo splicing assay was performed using the E1a or PTB minigene reporter (diagrams). (A) HeLa cells were cotransfected with the E1a reporter vector along with the mock vector or vector expressing a FLAG-tagged RBM4 homolog. Alternative splicing products were detected and quantified as in Figure 3. (B) HeLa cells were cotransfected with the PTB reporter and an RBM4 homolog expression vector as in panel A. RT-PCR was performed using total RNA as template and specific primers to PTB (Table 1). Numbers below the blot indicate the relative exon skipping efficiency (exon-skipped RNA/total RNA) of each RBM4 transfectant vs. mock (lane 1; mock was set to 1); average and standard deviation were obtained from three independent experiments. (C) Immunoblotting using anti-FLAG shows overexpressed FLAG-tagged Lark and RBM4 proteins as indicated. M: protein molecular size markers (kDa). D, Drosophila; B, Bombyx; C, Caenorhabditis; f, fish; m, mouse; h, human.
Figure 5.
The N-terminal RNA binding domain of human RBM4 dominates the splicing effect.
Diagram shows Drosophila Lark, zebrafish RBM4.1 and 4.2 and human RBM4a, and two chimeric proteins, RBM4-LC and RBM4-CP. RRM and CCHC represent RNA recognition motif and zinc knuckle, respectively. The in vivo splicing assay using the Kif1b reporter was performed as in Figure 3. Analysis of the splicing products was also as described in Figure 3. Relative efficiency for exon 25 inclusion was obtained from three independent experiments.
Figure 6.
Phosphorylation of RBM4 homologs by SRPK1.
In vitro phosphorylation of recombinant His-tagged RBM4 homologs and human Mago by GST-SRPK1 in the presence of [γ-32P]ATP. Proteins were fractionated by SDS-PAGE and detected by autoradiography (upper) and Coomassie blue staining (lower). Lane 1 shows the mock reaction. M: protein molecular size markers.
Figure 7.
Cellular localization of RBM4 homologs.
Top panels show endogenous (endo) RBM4: Double immunofluorescence was performed using anti-RBM4 and anti-SC35. To detect transiently expressed RBM4 orthologs, HeLa cells were transfected with a vector expressing FLAG-tagged human RBM4a, zebrafish RBM4.1 or RBM4.2, or Drosophila Lark, followed by immunofluorescence using anti-FLAG and anti-SC35. Cells were also stained with Hoechst 33258. Right panels show merged images. Scale bar, 8µm.
Table 1.
PCR Primers.