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Regulation and Function of Endocrine-Specific Splicing Programs in Pancreas and their Role in Diabetes

Periodic Reporting for period 1 - ePANC-SPLICE (Regulation and Function of Endocrine-Specific Splicing Programs in Pancreas and their Role in Diabetes)

Reporting period: 2020-01-01 to 2021-12-31

Diabetes affects ~10% of the adult population worldwide and its prevalence is rapidly increasing, representing a major health challenge. Diabetes is caused by dysfunction or loss of the insulin secreting beta cells of the pancreatic islets and the alteration of blood glucose levels. Despite the significant improvement of the life quality of diabetic patients during the last decades, there are currently no therapies to prevent or cure the disease. Thus, understanding how beta cells are generated, how they acquire their mature function and the ability to properly secrete insulin are major needs for the development of new therapies.
Extensive research in the field have uncovered the gene networks and signals that control the development of the different pancreatic endocrine cells. Moreover, the integration of genomics and genetics has revealed that genetic risk variants often alter the expression of key genes for beta cell function. Despite these remarkable advances, many aspects of gene regulation and function in pancreatic islets remain poorly understood. Among them, the roles of pre-mRNA alternative splicing (AS) have been largely unexplored. AS is a mechanism that allow single genes to produce multiple variants of its products (called RNAs and protein isoforms) by controlling the differential combination of gene pieces (introns and exons) into final molecules. This mechanism is a key generator of molecular diversity and plays pivotal roles in the development of organs and tissues, in the specialization of cell functions, and importantly, in several human diseases.
Thus, understanding how AS participates in beta cell differentiation and insulin secretory function, and its impact on glucose metabolism and diabetes can provide both basic and translatable knowledge for islet biology and for the development of novel therapies. Against this background, the goal of this project was to mechanistically and functionally characterize tissue-specific alternative splicing in endocrine pancreas, trying to answer the following questions: 1) Which are the master splicing regulators of AS in pancreas? 2) What is the impact of AS on beta cell development and secretory function? 3) Are endocrine-specific splicing programs deregulated in diabetes?
During the course of this project, we uncovered a conserved program of alternatively spliced microexons included specifically in islet cells. Our work revealed that islet microexons are regulated by the splicing factor SRRM3, and that dysregulation of the islet microexon program leads to defects in islet development and insulin secretion regulation, causing alteration of glucose homeostasis. Our findings thus provide novel insights into the transcriptional regulation of pancreatic endocrine cells development and function by alternative splicing.
The project had three main aims: 1) identify alternative splicing programs in pancreatic islet cells; 2) study their impact on beta cell function and their role in diabetes; 3) study their impact of on beta cell differentiation.
For the first aim, we collected RNAs-sequencing data of more than 50 human and rodent tissues and performed a computational analysis of transcriptome-wide alternative splicing profiles across tissues. Using this approach, we identified around 200 alternative exons specifically or preferentially included in RNAs of islet cells and excluded in the majority of other tissues. Strikingly, we found that half of these exons consisted in 3-27 nt short microexons (IsletMICs). We conducted silencing and overexpression experiments combined with RNAseq and identified the splicing factor SRRM3 as the master regulator of the IsletMIC program in endocrine pancreas.
For the second aim, we combined functional assays in cell lines with studies on a recently published mouse model. First, we combined insulin secretion experiments in beta cell lines, with different molecular and imaging techniques to study the activity of different process involved in insulin release. We found that depletion of SRRM3 leads to defective stimulated insulin release and that this is caused by the combination of altered cellular metabolism and changes in calcium signaling and cytoskeleton. To further study the role of microexons individually, we designed synthetic nucleic acid molecules to inhibit the inclusion of microexons. We found that misregulation of individual IsletMICs is sufficient to induce changes in glucose-stimulated insulin secretion. Next, to investigate the roles of IsletMICs in glucose metabolism in vivo, we took advantage of a recently published Srrm3 mutant mouse, kindly provided by Prof. Banfi (University of Iowa). RNA-seq analisis of isolated mouse islets confirmed the misregulation of IsletMIC splicing. Experiments in isolated mouse islets and measurements of blood glucose and insulin levels revealed that Srrm3 mutant mice present inappropriate insulin release and altered glucose homeostasis. Furthermore, we analyzed publicly available genetic and transcriptomic data of diabetic patients and found that the IsletMIC program is associated with risk of type 2 diabetes. Taken together, our observations indicate that the SRRM3-controlled microexon program plays essential roles in the regulation of insulin secretion and glucose homeostasis.
For the third aim, we combined developmental studies in zebrafish and mice with in vitro reprogramming to study the role of microexons during endocrine cell differentiation. We first studied islet development in a srrm3 mutant zebrafish line of developed in the lab. Mutant fishes presented alterations in the size and cellular composition of islets, and similar defects were also detected in Srrm3 mutant mice, indicating that srrm3 participates in late stages of endocrine pancreas development. To study the molecular mechanism of these defects, we have generated iPSC lines carrying a deletion of the SRRM3 gene and we will study how the lack of microexons affects their reprogramming towards pancreatic endocrine cells.
Over the last years, a plethora of studies have provided experimental evidence that alternative splicing play crucial roles in the development and function of a variety of cell and tissue types. However, its impact on endocrine pancreas and its physiological relevance in the context of glycemic control and disease remains largely unknown. In this project, we have identified a novel conserved program of islet-enriched alternative microexons and provide evidences that it plays important roles in establishing islet cell identity and function, globally impacting the regulation of glucose homeostasis and T2D susceptibility. These findings provide novel insights into endocrine pancreas regulation and the pathophysiology of diabetes that may in the future provide valuable knowledge for the development of novel therapeutic approaches. Improved understanding of alternative splicing in disease pathogenesis, together with advances in nucleic acid-based therapeutics, have rendered splicing modulation a promising therapeutic approach as evidenced with recent advances in Spinal Muscular Atrophy. In consequence, the discovery of splicing programs and events playing important roles in beta cell function is a necessary step to open new research avenues on splicing modulation in diabetes.