The RNA-binding profile of the splicing factor SRSF6 in immortalized human pancreatic β-cells

In pancreatic β-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes β-cell demise through splicing dysregulation of central genes for β-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic β-cell line EndoC-βH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in β-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic β-cells.Funding information: DL Eizirik is funded by Welbio/FRFS (no WELBIO-CR-2019C-04), Belgium; by the Brussels Region (INNOVIRIS BRIDGE grant DiaType), the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement numbers 115797 (INNODIA) and 945268 (INNODIA HARVEST); these Joint Undertakings receive support from the Union’s Horizon 2020 research and innovation program and “EFPIA” (European Federation of Pharmaceutical Industries Associations), “JDRF” (Juvenile Diabetes Research Foundation), “The Leona M and Harry B Helmsley Charitable Trust”), and the Dutch Diabetes Research Foundation (project Innovate2CureType1, DDRF; no. 2018.10.002). MI Alvelos was supported by Fonds pour la Formation a la Recherche dans l’Industrie et dans l’Agriculture (FRIA) fellowship from the Fonds de la Recherche Scientifique (FNRS) (reference no. 26410496), and COST: European Cooperation in Science & Technology (COST Action BM1207—Networking towards clinical application of antisense-mediated exon skipping; COST Action CA17103—Delivery of Antisense RNA Therapeutics). K Zarnack is funded by the German Research Foundation (DFG, SFB 902 – B13