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

Maria Inês Alvelos,Décio L Eizirik,Kathi Zarnack,Miguel Lopes,Ângela Castela,Fx Reymond Sutandy,Jonàs Juan-Mateu,Julian König,Anke Busch,Annemieke Aartsma-Rus,Maikel Luis Colli,Mirko Brüggemann

doi:10.26508/lsa.202000825

Abstract

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.

Highlights

Alternative splicing (AS) is a key co- and post-transcriptional mechanism regulating eukaryotic gene expression that determines which transcript and protein isoforms are formed under specific physiological and cellular contexts (Nilsen & Graveley, 2010; Liu et al, 2017)
By performing RNA sequencing (RNA-seq) following SRSF6 knockdown (KD) in EndoC-βH1 cells, we have previously shown that SRSF6 modulates the splicing of genes involved in different biological processes, such as β-cell survival, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling (Juan-Mateu et al, 2018)
To test whether AS can be modulated in EndoC-βH1 cells, we explored the application of antisense oligonucleotides (ASOs)

Summary

Introduction

Alternative splicing (AS) is a key co- and post-transcriptional mechanism regulating eukaryotic gene expression that determines which transcript and protein isoforms are formed under specific physiological and cellular contexts (Nilsen & Graveley, 2010; Liu et al, 2017). AS is regulated by RNA-binding proteins (RBPs) and their interactions with core spliceosomal components (Sanford et al, 2008; Wahl et al, 2009). RBPs bind to pre-mRNA binding sites that act as splicing-regulatory elements either enhancing or repressing the recognition of consensus splicing sequences and spliceosome assembly, thereby determining the final splicing outcome. Both spliceosomal components and RBPs, or in pre-mRNA sequences can lead to splicing dysregulation. Splicing alterations have been extensively studied in multiple tissues and splicing dysregulation has been increasingly recognized as a molecular mechanism associated with multiple human diseases (Braunschweig et al, 2013; Kelemen et al, 2013; Montes et al, 2019)

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