Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON Delta 165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.Institute of Molecular Biology Core Facilities; DFG [ZA 881/2-1, KO 4566/4-1, LE 3473/2-1]; LOEWE program Ubiquitin Networks (Ub-Net) of the State of Hesse (Germany); Deutsche Forschungsgemeinschaft [SFB902 B13]; EMBO [3057]; Fundacao para a Ciencia e a Tecnologia, Portugal (FCT Investigator Starting Grant) [IF/00595/2014]; German Federal Ministry of Research (BMBF; e:bio junior group program) [FKZ: 0316196]; Boehringer Ingelheim Foundation; [INST 47/870-1 FUGG

The prognostic potential of alternative transcript isoforms across human tumors

Background: Phenotypic changes during cancer progression are associated with alterations in gene expression, which can be exploited to build molecular signatures for tumor stage identification and prognosis. However, it is not yet known whether the relative abundance of transcript isoforms may be informative for clinical stage and survival. Methods: Using information theory and machine learning methods, we integrated RNA sequencing and clinical data from The Cancer Genome Atlas project to perform the first systematic analysis of the prognostic potential of transcript isoforms in 12 solid tumors to build new signatures for stage and prognosis. This study was also performed in breast tumors according to estrogen receptor (ER) status and melanoma tumors with proliferative and invasive phenotypes. Results: Transcript isoform signatures accurately separate early from late-stage groups and metastatic from non-metastatic tumors, and are predictive of the survival of patients with undetermined lymph node invasion or metastatic status. These signatures show similar, and sometimes better, accuracies compared with known gene expression signatures in retrospective data and are largely independent of gene expression changes. Furthermore, we show frequent transcript isoform changes in breast tumors according to ER status, and in melanoma tumors according to the invasive or proliferative phenotype, and derive accurate predictive models of stage and survival within each patient subgroup. Conclusions: Our analyses reveal new signatures based on transcript isoform abundances that characterize tumor phenotypes and their progression independently of gene expression. Transcript isoform signatures appear especially relevant to determine lymph node invasion and metastasis and may potentially contribute towards current strategies of precision cancer medicine.This work was supported by grants BIO2014-52566-R and Consolider RNAREG (CSD2009-00080) from the MINECO (Spanish Government) and FEDER, by AGAUR (2014-SGR1121) and by the Sandra Ibarra Foundation for Cancer (FSI2013)

Proneural and mesenchymal glioma stem cells display major differences in splicing and lncRNA profiles

Therapy resistance and recurrence in high-grade gliomas are driven by their populations of glioma stem cells (GSCs). Thus, detailed molecular characterization of GSCs is needed to develop more effective therapies. We conducted a study to identify differences in the splicing profile and expression of long non-coding RNAs in proneural and mesenchymal GSC cell lines. Genes related to cell cycle, DNA repair, cilium assembly, and splicing showed the most differences between GSC subgroups. We also identified genes distinctly associated with survival among patients of mesenchymal or proneural subgroups. We determined that multiple long non-coding RNAs with increased expression in mesenchymal GSCs are associated with poor survival of glioblastoma patients. In summary, our study established critical differences between proneural and mesenchymal GSCs in splicing profiles and expression of long non-coding RNA. These splicing isoforms and lncRNA signatures may contribute to the uniqueness of GSC subgroups, thus contributing to cancer phenotypes and explaining differences in therapeutic responses.This study was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil to PAFG and LOFP and by NIH 7R21CA175875-03. This study was partially supported by grants from Serrapilheira foundation and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2018/15579-8) to PAFG. GDAG and BRC were supported by fellowships from FAPESP (2017/19541-2) and (2013/25483-4 and 2013/07159-5), respectively. PRA was supported by CPRIT Training Grant - RP14010

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection

RNA splicing factors as oncoproteins and tumour suppressors

The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutationsclos