The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

Human Genetics of Atrial Septal Defect.

Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.

RBM10 C761Y mutation induced oncogenic ASPM isoforms and regulated ß-catenin signaling in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) comprises a heterogeneous group of biliary tract cancer. Our previous CCA mutation pattern study focused on genes in the post-transcription modification process, among which the alternative splicing factor RBM10 captured our attention. However, the roles of RBM10 wild type and mutations in CCA remain unclear. METHODS: RBM10 mutation spectrum in CCA was clarified using our initial data and other CCA genomic datasets from domestic and international sources. Real-time PCR and tissue microarray were used to detect RBM10 clinical association. Function assays were conducted to investigate the effects of RBM10 wild type and mutations on CCA. RNA sequencing was to investigate the changes in alternative splicing events in the mutation group compared to the wild-type group. Minigene splicing reporter and interaction assays were performed to elucidate the mechanism of mutation influence on alternative splicing events. RESULTS: RBM10 mutations were more common in Chinese CCA populations and exhibited more protein truncation variants. RBM10 exerted a tumor suppressive effect in CCA and correlated with favorable prognosis of CCA patients. The overexpression of wild-type RBM10 enhanced the ASPM exon18 exon skipping event interacting with SRSF2. The C761Y mutation in the C2H2-type zinc finger domain impaired its interaction with SRSF2, resulting in a loss-of-function mutation. Elevated ASPM203 stabilized DVL2 and enhanced ß-catenin signaling, which promoted CCA progression. CONCLUSIONS: Our results showed that RBM10C761Y-modulated ASPM203 promoted CCA progression in a Wnt/ß-catenin signaling-dependent manner. This study may enhance the understanding of the regulatory mechanisms that link mutation-altering splicing variants to CCA.

RBM10 Mutation as a Potential Negative Prognostic/Predictive Biomarker to Therapy in Non-Small-Cell Lung Cancer.

BACKGROUND: According to WHO, lung cancer is the leading cause of cancer-related death worldwide, but treatment has advanced in the last decade. The widespread use of Next Generation Sequencing has led to the discovery of several pathogenic mutations including RNA binding motif 10 (RBM10), a part of the spliceosome complex that regulates splicing of pre-mRNA. PATIENTS AND METHODS: Electronic medical records were utilized to create a database of patients (50 patients) seen from 2018-2023 with NSCLC and RBM10 mutations, with appropriate IRB approval. For subgroup analysis, we separated into groups by rapid progression vs stable disease defined as progression-free survival earlier than respective clinical trials. RESULTS: From the analysis of treatment response the mutated RBM10 population had a median PFS was 6.7 months compared to 13.9 in the wild-type RBM10 population controlled for driver mutations TP53 mutation had a higher representation in the RBM10 mutated rapid progression group than the stable disease group. The ZFHX3 mutation had a higher representation in the RBM10 mutated stable disease group. CONCLUSIONS: RBM10 mutations were associated with aggressive disease with treatment progression faster than median durations of response. RBM10 mutations with concurrent ZFHX3 and EGFR mutations were associated with more stable disease, while concurrent KRAS and TP53 predicted even more aggressive disease.

Down regulation of RBM10 promotes proliferation and metastasis via miR-224-5p/RBM10/p53 feedback loop in lung adenocarcinoma.

RNA-binding motif protein 10 (RBM10) has a tumor suppressor role in multiple cancers. Combining Oncomine database results with tissue samples, Western blot analysis showed that RBM10 was significantly lower in lung adenocarcinoma (LUAD) than in adjacent normal tissues. Moreover, KM analysis revealed that the group with higher RBM10 expression in LUAD correlated with better overall survival (OS). Luciferase reporter assay revealed that an important tumor-promotive miRNA, miR-224-5p, was directly bound to the 3'UTR of RBM10, resulting in inhibition of RBM10 expression, and promoted LUAD progression both in vitro and in vivo. Mechanistically, we found that miR-224-5p directly targeted RBM10 to inhibit p53 expression during LUAD progression. Meanwhile, p53 affected RBM10 expression through p53/miR-224-5p axis. Our study identified RBM10 as a key tumor suppressor in the proliferation and metastasis of LUAD. The findings provide a novel mechanism involving a feedback loop of miR-224-5p/RBM10/p53 regulated tumor progression in LUAD, which may help with the design of more effective LUAD treatments.

Investigation of RBM10 mutation and its associations with clinical and molecular characteristics in EGFR-mutant and EGFR-wildtype lung adenocarcinoma.

Background: RBM10 is commonly mutated in lung adenocarcinoma (LUAD). However, its role in the pathogenesis of LUAD remains undefined. EGFR-mutant LUAD represents a distinct subset of non-small cell lung cancer (NSCLC). The function of RBM10 in tumor pathogenesis is supposed to differ between EGFR-mutant and EGFR-wt LUAD. This study aimed to interrogate the prevalence of RBM10 mutation in a large cohort of Chinese patients with LUAD and investigate the association of RBM10 mutation with clinical and molecular characteristics of EGFR-mutant and EGFR-wt LUAD. Methods: Tumor sequencing data from 2848 Chinese patients with LUAD were retrospectively reviewed and analyzed. The prevalence of RBM10 was also compared with other three cohorts: OrigMed (n = 1222), MSKCC (n = 1267), and TCGA (n = 566). The associations of RBM10 mutation with clinical and molecular characteristics were assessed. An external cohort of 182 patients with LUAD who received PD-1 inhibitor were used to investigate the association of RBM10 mutation with clinical outcomes upon immunotherapy. Results: Our cohort showed a higher prevalence of RBM10 in EGFR-mutant LUAD than in EGFR-wt LUAD (14.8 % vs. 6.5 %, p < 0.001). The enrichment of RBM10 mutations in EGFR-mutant LUAD was also seen in another Chinese cohort (OrigMed: 14.9 % vs. 7.8 %, p < 0.001), but not in the two western cohorts (MSKCC: 7.4 % vs. 9.5 %, p = 0.272; TCGA: 8.1 % vs. 6.7 %, p = 0.624). RBM10 mutations co-occurred more frequently with EGFR L858R mutations (23.7 %) than with other types of EGFR mutations (19 del: 7.7 %; other: 7.1 % in others, p < 0.001). In EGFR-mutant LUAD, RBM10 mutations were more commonly found in stage I (18.2 %) and II (21.8 %) vs. stage III (9.4 %) and IV (11.3 %) tumors (p < 0.001). The proportion of PD-L1 positive expression in EGFR-mutant LUAD with concomitant RBM10 mutation was not different from that those without RBM10 mutations (41.8 % vs. 47.9 %, p = 0.566). In contrast, RBM10 mutation occurred more frequently in EGFR-wt LUAD at stage II-IV (stage II: 12.0 %, stage III: 8.7 %, stage IV: 6.6 %) than at stage I (2.8 %). EGFR-wt LUAD with concomitant RBM10 mutations had higher proportions of PD-L1 expression positivity (78.9 % vs. 61.9 %, p = 0.014) and higher tumor mutational load (8.97 vs. 2.99 muts/Mb, p < 0.001) than those without. Patients with EGFR-wt LUAD who also harbored RBM10 loss of function (LOF) mutations had a longer median PFS upon immunotherapy than those with RBM10 non-LOF mutations (7.15 m vs. 2.60 m, HR = 4.83 [1.30-17.94], p = 0.010). Conclusion: We comprehensively investigated RBM10 mutations in a Chinese cohort with LUAD. Compared to western cohorts, a significant enrichment of RBM10 mutations in EGFR-mutant LUAD compared to EGFR-wildtype LUAD in the Chinese population. RBM10 mutation shows different associations with clinical and molecular characteristics between EGFR-mutant and EGFR-wt LUAD, suggesting a divergent mechanism between these two subsets via which RBM10 deficiency contribute to tumor pathogenesis. The findings contribute to our understanding of the molecular landscape of LUAD and highlight the importance of considering population-specific factors in cancer genomics research.

Proto-oncogene cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program in the heart and controls cardiac hypertrophy.

AIMS: RBM10 is a well-known RNA binding protein that regulates alternative splicing in various disease states. We have shown a splicing-independent function of RBM10 that regulates heart failure. This study aims to unravel a new biological function of RBM10 phosphorylation by proto-oncogene cSrc that enables anti-hypertrophy gene program and controls cardiac hypertrophy. MATERIALS AND METHODS: We employ in vitro and in vivo approaches to characterise RBM10 phosphorylation at three-tyrosine residues (Y81, Y500, and Y971) by cSrc and target mRNA regulation. We also use isoproterenol induced rat heart and cellular hypertrophy model to determine role of cSrc-mediated RBM10 phosphorylation. KEY FINDINGS: We show that RBM10 phosphorylation is induced in cellular and animal heart model of cardiac hypertrophy and regulates target mRNA expression and 3'-end formation. Inhibition of cSrc kinase or mutation of the three-tyrosine phosphorylation sites to phenylalanine accentuates myocyte hypertrophy, and results in advancement and an early attainment of hypertrophy in the heart. RBM10 is down regulated in the hypertrophic myocyte and that its re-expression reverses cellular and molecular changes in the myocyte. However, in the absence of phosphorylation (cSrc inhibition or phospho-deficient mutation), restoration of endogenous RBM10 level in the hypertrophic heart or ectopic re-expression in vitro failed to reverse cardiomyocyte hypertrophy. Mechanistically, loss of RBM10 phosphorylation inhibits nuclear localisation and interaction with Star-PAP compromising anti-hypertrophy gene expression. SIGNIFICANCE: Our study establishes that cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program. We also report a new functional regulation of RBM10 by phosphorylation that is poised to control heart failure.

RBM10 loss induces aberrant splicing of cytoskeletal and extracellular matrix mRNAs and promotes metastatic fitness.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse Hras G12V /Rbm1O KO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.