Periodic Reporting for period 1 - REMIX (Effects of rewiring microexons (REMIX) on tissue-specific signaling networks) Reporting period: 2018-03-01 to 2020-02-29 Summary of the context and overall objectives of the project Alternative splicing of messenger RNA in different tissues of the body allows custom-tailoring the function of a given gene to its environment. This mechanism of increasing genetic diversity is also frequently hijacked by tumors to improve evolutionary fitness, by selecting for splice variants that provide a growth and/or survival advantage to the cell. For example, signaling pathways that promote inappropriate proliferative activity can be upregulated through the selective use of isoforms with increased activity. In this MSC action, we focused on a specific type of alternative splicing called microexon splicing, and we aimed to understand the role it plays in regulating the activity of kinases, one of the key effectors of oncogenic signaling pathways. By calculating a tissue-specificity score for all kinase microexons, we determined that microexons outside of the catalytic kinase domain are significantly more likely to display alternative splicing patterns across different tissues than those within the kinase domain, which generally display constitutive splicing patterns. This finding suggests that microexons in kinases serve to modulate their interactions with other proteins, rather than altering their catalytic activity or substrate specificity at the peptide level. Analysis of transcriptomic data from The Cancer Genome Atlas revealed a global downregulation of microexon inclusion in tumors, driven by hypersilencing of the microexon splicing factor SRRM4. This discovery revealed a previously uncovered antiproliferative role for microexons and SRRM4 in non-neural tissues, adding a new element to the overall picture of splicing dysregulation in cancer. These findings are important for society as they help reveal how changes to RNA molecules promote tumor growth, which is important for identifying novel cancer treatment strategies. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this MSC Action, the overall goal was to perform the first systematic study of microexons in kinases, to uncover their specific functions in controlling tissue-specific signaling networks and determine whether dysregulation of microexon inclusion is involved in cancer pathogenesis. The work performed during the course of this project revealed that a majority of microexons located within the kinase domain do not display tissue-specific inclusion patterns, suggesting that these exons are not major contributors to tissue-specific rewiring of signaling networks. Instead, kinase microexons outside of the kinase domain itself appear to be more likely candidates for rewiring microexons. Our analysis of microexon inclusion using patient data from The Cancer Genome Atlas revealed a global downregulation of microexons in tumors across tissue types, resulting from hypersilencing of the microexon splicing factor SRRM4. This finding represents a paradigm shift in the microexon field, as the vast majority of research on microexons has focused exclusively on their roles in the brain. In contrast, our findings suggest an antiproliferative role of microexons in non-neural tissues, leading to a selective pressure for decreased microexon inclusion during tumor evolution. The scientific results arising from this project will be disseminated through two scientific journal articles, one under revision and one in preparation, which upon publication will be promoted through CRG social media channels. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The results obtained during the course of this action will have impact for researchers of alternative splicing in signaling proteins and in the cancer research field. Previous studies of alternative splicing alterations in cancer have not specifically taken microexons into account, due to the technical challenges presented by their detection as well as a longstanding underappreciation of their biological relevance. As such, the finding that SRRM4 and its microexon splicing program are silenced in tumors contributes an important new element to the overall picture of splicing dysregulation in cancer. Our analysis of splicing changes using TCGA data has already been made publicly available in advance of the publication of our first article, allowing other researchers in the field to benefit from our findings as early as possible. Other data generated during the course of this project will be used to apply for further sources of research funding, amplifying the future impact of the action. Furthermore, our discoveries may provide novel opportunities for therapeutic intervention in a wide range of cancers. With the advent of splice-switching antisense oligonucleotides that are now being used therapeutically to affect exon inclusion levels, this knowledge could guide the development of new strategies for cancer treatment. Ongoing work seeks to identify specific microexons that could be targeted therapeutically, which could have a large impact on society if novel treatments are developed based on this work. In addition, the publication of our work in scientific journals will help to improve international visibility of the CRG and Spanish and European research and foster future collaborations. Finally, the action will have a great impact on the future career prospects of the researcher, through the achieved scientific productivity, scientific and career-focused training, competitiveness for future funding opportunities and international mobility taken advantage of during the action. Graphical abstract of study results
