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Periodic Reporting for period 1 - Beta-splicenet (ALTERNATIVE SPLICING NETWORKS IN PANCREATIC BETA CELLS)

Reporting period: 2015-06-01 to 2017-05-31

The project Beta-splicenet focused in studying the role of RNA alternative splicing (AS), a complex mechanism of gene regulation, in insulin-producing pancreatic beta cells. Our goal was to understand how AS regulates beta cell function and survival, and how alterations in this process may contribute to the development of type 1 diabetes (T1D). T1D results from the interaction between predisposing genes and environmental factors, triggering an autoimmune attack against pancreatic beta cells that leads to their progressive loss due to cell death. However, the molecular mechanisms and signals promoting beta cell dysfunction and loss are poorly understood. AS allows single genes to produce multiple protein variants with different functions, playing a critical role in the control of cell-specific functions. In this project, we used a systems biology approach combining next generation sequencing, bioinformatics and functional studies in beta cell models, to unveil splicing networks that regulate key beta cell functions that may be altered during the development of T1D. Moreover, we also explored the modulation of AS by small molecules as a novel therapeutic approach to prevent beta cell loss. During this project we identified several splicing regulators and their associated RNA networks that modulate the activity of key pathways and processes for the survival and secretory function of beta cells. Moreover, we showed that some of these splicing networks are dysregulated by inflammation or diabetes susceptibility genes, contributing to the general beta cell demise that occurs during the development of the disease.
1. Prediction of splicing regulatory networks from RNA-seq data.
We performed RNA-sequencing of human islets and human insulin-producing EndoC-βH1 cells exposed to pro-inflammatory cytokines, as a T1D in vitro model. Bioinformatics analysis lead to the identification of thousands of alternative splicing (AS) events affected by inflammation. These events affect important molecular pathways for the correct function and survival of beta cells, including cell death regulation, insulin secretion and stress-activated pathways. We used sequence motif enrichment analysis to scan alternatively spliced genes and detect candidate RNA-binding proteins (RBPs) regulators. Integration of these data with gene expression values enabled us to predict splicing regulatory networks.
2: Validation of candidate splicing networks and functional assessment in beta cell models.
Some networks predicted in aim 1 were selected for further functional studies in in vitro beta cell models. We identified a group of RBPs that are preferentially expressed in beta cells and brain. We selected four RBPs, namely Elavl4, Nova2, Rbox1 and Rbfox2, for subsequent functional studies in rodent and human beta cells. We found that inhibition of Elavl4 and Nova2 increased beta cell death, while silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, we found that Rbfox1 silencing modulates the splicing of calcium channels and actin-remodelling proteins, leading to increased calcium influx and faster glucose-induced actin depolymerisation and thus enhancing the insulin secretory capacity. These findings indicate that beta cells share common splicing programs with neurons that play key roles for insulin release and beta cell survival.
We also found that SRp55 is key splicing regulator of human beta cells. SRp55 expression is regulated by the T1D and T2D susceptibility gene GLIS3 and by cytokines suggesting a direct role in diabetes. We found that SRp55 silencing increases beta cell death, impairs insulin secretion and induces endoplasmic reticulum stress. RNA-sequencing of SRp55-depleted EndoC-βH1 cells allowed to identify hundreds of previously unknown SRp55-regulated splicing events. SRp55 deficiency impacts on crucial pathways for beta cell survival and function. Follow up mechanistic experiments showed that SRp55 depletion affects cell death regulatory proteins and stress-activated signalling pathways, leading to increased beta cell fragility and death. Moreover, SRp55 regulates genes controlling insulin secretion and defining beta cell identity. Our findings indicate that SRp55 is a master splicing factor of beta cells and a key down-stream mediator of GLIS3 function.
3: Identification of cis-elements regulating candidate mRNA variants and modulation by ASO targeting.
Two alternatively spliced genes were selected for a splicing modulation approach. Regulators of beta cell death BIM and BAX were cloned into mini-gene plasmids to develop a splicing reporter assay that recapitulates the observed splicing change and allows, by site-directed mutagenesis, to screen for splicing regulatory motifs. Using AS, these genes are able to produce several variants with different pro-death activities. Our goal was to identify specific RNA sequences that control splicing and target them using antisense oligonucleotides (ASO) to improve beta cell survival. We identified different regions in the BIM pre-mRNA that have a positive or negative impact on the expression of the most pro-death variant BIM S. We developed an ASO against an exonic region that proved to be effective in decreasing the expression of BIM S in cytokine-treated EndoC-βH1 beta cells. The potential protective effect of this ASO on immune-induced beta cell death is currently been tested in different beta cell models.

The results generated during this project have been published in specialized scientific journals and presented in different international scientific conferences in Europe and in the USA. Moreover, dissemination of the knowledge produced by this project for the general public was performed through publication of educational articles and by presentations in universities and secondary schools.
There has been a growing interest in understanding the role of alternative splicing in the regulation of cell-specific functions and disease, but almost nothing was known about its role in pancreatic beta cell and diabetes. The prevalence of T1D is doubling every 25 years and there are presently no adequate approaches to prevent or cure the disease. During this project, we have shown that dysregulation of specific splicing networks have a major impact on beta cell survival, insulin secretion and maintenance of the beta cell differentiated phenotype. We identified key splicing regulators and networks that are modulated by inflammation or diabetes susceptibility genes, and thus contribute to the functional impairment and death of beta cells. Our results unveil a new level of gene regulation in beta cells that probably contributes to beta cell fragility and lost during T1D. Our studies also identified potential therapeutic RNA targets, opening new avenues for therapeutic intervention based on splicing modulatory drugs. Following this idea, we designed a small molecule targeting a regulator of beta cell death, aiming to modulate its splicing and improve beta cell survival. Preliminary results are promising but follow-up studies are needed to evaluate the potential applicability of this approach. In conclusion, we believe that the Beta-splicenet project has generated important knowledge for both the scientific community and the general society, helping to understand the pathogenesis of T1D and exploring new therapeutic strategies.