Martin Dutertre

Cotranscriptional exon skipping in the genotoxic stress response

Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin (CPT), inhibit the interaction between Ewings sarcoma proto-oncoprotein (EWS), an RNA polymerase II–associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene, which encodes the main p53 ubiquitin ligase. This reversible exon skipping participates in the regulation of MDM2 expression that may contribute to the accumulation of p53 during stress exposure and its rapid shut-off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to CPT and EWS–YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, which results in widespread exon skipping and plays a central role in the genotoxic stress response.

Splicing factor and exon profiling across human tissues

It has been shown that alternative splicing is especially prevalent in brain and testis when compared to other tissues. To test whether there is a specific propensity of these tissues to generate splicing variants, we used a single source of high-density microarray data to perform both splicing factor and exon expression profiling across 11 normal human tissues. Paired comparisons between tissues and an original exon-based statistical group analysis demonstrated after extensive RT-PCR validation that the cerebellum, testis, and spleen had the largest proportion of differentially expressed alternative exons. Variations at the exon level correlated with a larger number of splicing factors being expressed at a high level in the cerebellum, testis and spleen than in other tissues. However, this splicing factor expression profile was similar to a more global gene expression pattern as a larger number of genes had a high expression level in the cerebellum, testis and spleen. In addition to providing a unique resource on expression profiling of alternative splicing variants and splicing factors across human tissues, this study demonstrates that the higher prevalence of alternative splicing in a subset of tissues originates from the larger number of genes, including splicing factors, being expressed than in other tissues.

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss. Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3ss-sequence context. SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

A Subset of Nuclear Receptor Coregulators Act as Coupling Proteins during Synthesis and Maturation of RNA Transcripts

Gene expression is a multistep process starting in the cell nucleus with the synthesis of the primary transcripts that undergo several modifications (including capping, splicing, and polyadenylation) leading to the export of the mature mRNAs into the cytoplasm for translation into proteins. Although an emerging view is that all the steps from transcription to translation are mechanically and functionally coupled, the proteins that are involved in this coupled process are still poorly characterized. In recent years, a growing list of proteins known to control gene expression at the transcriptional level, named transcriptional coregulators, have been independently shown to play additional roles in other steps of gene expression. In this review we compile these emerging data suggesting that a subset of transcriptional coregulators play a major role in the coordination of the individual steps of the gene expression process.

Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.

FAST DB: a website resource for the study of the expression regulation of human gene products

Human genes use various mechanisms to generate different transcripts having different exon content, which in turn generate multiple protein isoforms having differential and even opposite biological activities. To understand the biological consequences of gene transcriptional activity modulation, it is necessary to integrate the capability of genes to generate distinct functional products, particularly because transcriptional stimuli also affect the exon content of their target gene products. For this purpose, we have developed a bioinformatics suite, FAST DB, which defines easily and accurately the exon content of all known transcripts produced by human genes. In addition, several tools have been developed, including a graphical presentation of all gene products, a sequence multi-alignment of all gene transcripts and an in silico PCR computer program. The FAST DB interface also offers extensive links to website resources for promoter analysis and transcription factor binding site prediction, splicing regulatory sequence prediction, as well as 5′- and 3′-untranslated region analysis. FAST DB has been designed to facilitate studies that integrate transcriptional and post-transcriptional events to investigate the expression regulation of human gene products.

Splicing switch of an epigenetic regulator by RNA helicases promotes tumor-cell invasiveness

Both epigenetic and splicing regulation contribute to tumor progression, but the potential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases Ddx17 and Ddx5 contribute to tumor-cell invasiveness by regulating alternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an opposite manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone variant isoforms that in turn drive a transcription program to switch tumor cells to an invasive phenotype.

A new advance in alternative splicing databases: from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants

BackgroundMost human genes produce several transcripts with different exon contents by using alternative promoters, alternative polyadenylation sites and alternative splice sites. Much effort has been devoted to describing known gene transcripts through the development of numerous databases. Nevertheless, owing to the diversity of the transcriptome, there is a need for interactive databases that provide information about the potential function of each splicing variant, as well as its expression pattern.DescriptionAfter setting up a database in which human and mouse splicing variants were compiled, we developed tools (1) to predict the production of protein isoforms from these transcripts, taking account of the presence of open reading frames and mechanisms that could potentially eliminate transcripts and/or inhibit their translation, i.e. nonsense-mediated mRNA decay and microRNAs; (2) to support studies of the regulation of transcript expression at multiple levels, including transcription and splicing, particularly in terms of tissue specificity; and (3) to assist in experimental analysis of the expression of splicing variants. Importantly, analyses of all features from transcript metabolism to functional protein domains were integrated in a highly interactive, user-friendly web interface that allows the functional and regulatory features of gene transcripts to be assessed rapidly and accurately.ConclusionIn addition to identifying the transcripts produced by human and mouse genes, fast DB http://www.fast-db.com provides tools for analyzing the putative functions of these transcripts and the regulation of their expression. Therefore, fast DB has achieved an advance in alternative splicing databases by providing resources for the functional interpretation of splicing variants for the human and mouse genomes. Because gene expression studies are increasingly employed in clinical analyses, our web interface has been designed to be as user-friendly as possible and to be readily searchable and intelligible at a glance by the whole biomedical community.

Exon-Based Clustering of Murine Breast Tumor Transcriptomes Reveals Alternative Exons Whose Expression Is Associated with Metastasis

In the field of bioinformatics, exon profiling is a developing area of disease-associated transcriptome analysis. In this study, we performed a microarray-based transcriptome analysis at the single exon level in mouse 4T1 primary mammary tumors with different metastatic capabilities. A novel bioinformatics platform was developed that identified 679 genes with differentially expressed exons in 4T1 tumors, many of which were involved in cell morphology and movement. Of 152 alternative exons tested by reverse transcription-PCR, 97 were validated as differentially expressed in primary tumors with different metastatic capability. This analysis revealed candidate progression genes, hinting at variations in protein functions by alternate exon usage. In a parallel effort, we developed a novel exon-based clustering analysis and identified alternative exons in tumor transcriptomes that were associated with dissemination of primary tumor cells to sites of pulmonary metastasis. This analysis also revealed that the splicing events identified by comparing primary tumors were not aberrant events. Lastly, we found that a subset of differentially spliced variant transcripts identified in the murine model was associated with poor prognosis in a large clinical cohort of patients with breast cancer. Our findings illustrate the utility of exon profiling to define novel theranostic markers for study in cancer progression and metastasis.

The emerging role of pre-messenger RNA splicing in stress responses: Sending alternative messages and silent messengers

Alternative splicing (AS) of pre-messenger RNAs is a major process contributing to both transcriptome and proteome diversity in various physiological and pathological situations. There is also accumulating evidence that various stresses impact on AS. In particular, recent analyses of the transcriptome reveal large numbers of AS events that are regulated by genotoxic stress inducers like radiations and chemotherapeutic agents. Many AS events have the potential to affect the relative production of protein isoforms with different activities, as shown in the case of several genes involved in apoptosis. There is also increasing evidence that stresses induce “non-productive” splice variants, leading to a decrease in gene expression levels or preventing increases in protein levels despite transcriptional stimulation. This is typically achieved by the production of splice variants that are subject to nonsense-mediated decay. In addition, recent studies suggest that pre-mRNA splicing efficiency or fidelity may be altered by stresses. For example, various genotoxic agents induce multiple exon skipping in MDM2 transcripts, thereby preventing the production of the main p53-ubiquitin ligase and favoring p53 activity in response to genotoxic agents. In terms of mechanisms, stresses can impact on pre-mRNA splicing by inducing post-translational modifications and subcellular redistribution of splicing factors, or by targeting the communication between the splicing and transcription machineries. Altogether, these data suggest that splicing regulatory networks play a key role in the cellular responses triggered by stresses.