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
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.
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.