RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation

Abstract

In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.

Keywords: RNA splicing; alternative splicing; protein methylation; protein-protein interaction; proteomics.

Figure 1.

RBM25 is methylated in human cells and required for cell growth in culture. A, RBM25 was immunoprecipitated from HT-1080 and 293T cells, and Lys-77 methylation was measured by in-gel digestion with chymotrypsin followed by LC/MS-MS. Chromatographs show the relative intensity of non-methyl and mono-methylated forms of the peptide containing lysine 77 (amino acids 75–90, KAKENDENC*GPTTTVF, non-methyl and mono-methyl monitored at m/z 604.2807 and 608.9526, respectively). The asterisk indicates carbamidomethylation. B, RBM25 levels in 293T cells were measured by Western blot 4 days after transduction with the CRISPR/Cas9 system. C, growth of transduced 293T cells was tracked for 8 days either with puromycin selection (left panel) or without selection (right panel). Error bars indicate mean ± S.E. (n = 3). D and E, B and C repeated in HT-1080 cells. F, RBM25 knockdown and reconstitution in 293T cells was measured by Western blotting with near-IR fluorescent secondary antibodies. Densitometry indicates RBM25 expression relative to endogenous, normalized to β-tubulin. G, 293T cells expressing exogenous RBM25 or GFP (negative control) were transduced with CRISPR/Cas9 and sgRNA against RBM25 or a non-targeting control. Cells were passaged every 2 days and counted 8 days after transduction (S.E., n = 4, Student's t test). H and I, F and G repeated in HT-1080 cells. J, cell cycle distribution of 293T cells was measured by propidium iodide stain and flow cytometry 4 days after transduction with CRISPR/Cas9. Values are the average of three replicates.

Figure 2.

RBM25 is a global regulator of transcription and alternatively spliced exons. A, RNA-Seq was used to identify genes affected by RBM25 knockdown relative to non-targeting sgRNA. Enriched GO terms were identified using DAVID (Benjamini-Hochberg–corrected p < 0.05). B, splice events regulated by RBM25 knockdown were identified by processing sequencing data with JuncBASE and applying thresholds for 5% FDR and a minimum 10% change in use of the exon or alternative splicing event (e.g. 80% of transcripts to 70% of transcripts). C, endogenous RBM25 was depleted in 293T cells expressing a control vector (GFP) or RBM25, as shown in Fig. 1E. Real-time PCR was used to measure exon inclusion at eight RBM25 target exons identified by RNA-Seq (four shown here and four shown in supplemental Fig. S2). Error bars show S.E., n = 4. D, analysis of motif enrichment (25) was used to compare the frequency of motifs resembling GGGGGGG in exons positively regulated by RBM25 to a background of alternatively spliced exons. E, overlap of differentially expressed genes and genes containing exons positively regulated by RBM25. Statistical significance of the overlap was calculated using Fisher's exact test.

Figure 3.

RBM25 interacts with splicing machinery through several protein domains. A, quantitative LC/MS-MS using SILAC was used to compare proteins bound by RBM25 co-IP relative to IgG control. DAVID was used to determine GO terms enriched among RBM25-interacting proteins (selected terms are shown, p < 10⁻⁴⁰). B, the STRING protein interaction database was used to visualize the RBM25 interactome. Selected cliques of interacting protein families or complexes are shown. C, RBM25 lacking specific domains were expressed with an N-terminal FLAG tag, and FLAG co-IP with quantitative LC/MS-MS with SILAC was used to measure the effect on protein interactions. D–F, the effect of each domain deletion was measured in duplicate with isotopic labels reversed. Bar plots show the average amount of protein bound by the short form relative to the long form of RBM25 (all normalized to a molar quantity of RBM25), and error bars show high and low values for each pair of measurements. The light gray horizontal line indicates 1:1 binding. Selected protein families are shown.

Figure 4.

SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A, immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B, relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log₂ scale. Several SR proteins are highlighted in red. C, recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D, SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E, RBM25 and SRSF2 were expressed by transient transfection in 293T cells with FLAG and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F, a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G, SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary antibodies. Error bars indicate mean ± S.E. (n = 3).