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

Alvelos, Maria Inês; Brüggemann, Mirko; Sutandy, F. X. Reymond; Juan-Mateu, Jonàs; Colli, Máikel Luís; Busch, Anke; Lopes, Miguel; Castela, Ângela; Aartsma‐Rus, Annemieke; König, Julian; Zarnack, Kathi; Eizirik, Décio L.

doi:10.26508/lsa.202000825

Cited by 17 publications

(27 citation statements)

References 120 publications

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“…The function of these different protein products can vary greatly and impact cellular function ( Alvelos et al 2018 ). Recent studies have suggested the importance of the unique splicing program innate to pro-endocrine cells, and how the functional outputs help steer the developmental process, yet it remains incompletely understood ( Juan-Mateu et al 2018 ; Singer et al 2019 ; Colli et al 2020 ; Alvelos et al 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs

Dudek

Osipovich

Cartailler

et al. 2021

G3 Genes|Genomes|Genetics

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Insm1, Neurod1, and Pax6 are essential for the formation and function of pancreatic endocrine cells. Here, we report comparative immunohistochemical, transcriptomic, functional enrichment, and RNA splicing analyses of these genes using gene knock-out mice. Quantitative immunohistochemical analysis confirmed that elimination of each of these three factors variably impairs the proliferation, survival, and differentiation of endocrine cells. Transcriptomic analysis revealed that each factor contributes uniquely to the transcriptome although their effects were overlapping. Functional enrichment analysis revealed that genes downregulated by the elimination of Insm1, Neurod1, and Pax6 are commonly involved in mRNA metabolism, chromatin organization, secretion, and cell cycle regulation, and upregulated genes are associated with protein degradation, autophagy, and apoptotic process. Elimination of Insm1, Neurod1, and Pax6 impaired expression of many RNA-binding proteins thereby altering RNA splicing events, including for Syt14 and Snap25, two genes required for insulin secretion. All three factors are necessary for normal splicing of Syt14, and both Insm1 and Pax6 are necessary for the processing of Snap25. Collectively, these data provide new insights into how Insm1, Neurod1, and Pax6 contribute to the formation of functional pancreatic endocrine cells.

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Section: Discussionmentioning

confidence: 99%

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs

Dudek

Osipovich

Cartailler

et al. 2021

G3 Genes|Genomes|Genetics

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“…Inhibition of the candidate gene GLIS3 has a proapoptotic effect on human b-cells, which is mediated at least in part via downregulation of the splicing factor SRSF6 (also known as SRp55). SRSF6 inhibition impairs directly or indirectly the splicing of pre-mRNAs encoding key proteins for b-cell function (e.g., INSR, SNAP25), and survival (e.g., BIM-Small, BAX-b) (41,44).…”

Section: The As Connectionmentioning

confidence: 99%

From Pancreatic β-Cell Gene Networks to Novel Therapies for Type 1 Diabetes

et al. 2021

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Completion of the Human Genome Project enabled a novel systems- and network-level understanding of biology, but this remains to be applied for understanding the pathogenesis of type 1 diabetes (T1D). We propose that defining the key gene regulatory networks that drive β-cell dysfunction and death in T1D might enable the design of therapies that target the core disease mechanism, namely, the progressive loss of pancreatic β-cells. Indeed, many successful drugs do not directly target individual disease genes but, rather, modulate the consequences of defective steps, targeting proteins located one or two steps downstream. If we transpose this to the T1D situation, it makes sense to target the pathways that modulate the β-cell responses to the immune assault—in relation to signals that may stimulate the immune response (e.g., HLA class I and chemokine overexpression and/or neoantigen expression) or inhibit the invading immune cells (e.g., PDL1 and HLA-E expression)—instead of targeting only the immune system, as it is usually proposed. Here we discuss the importance of a focus on β-cells in T1D, lessons learned from other autoimmune diseases, the “alternative splicing connection,” data mining, and drug repurposing to protect β-cells in T1D and then some of the initial candidates under testing for β-cell protection.

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“…First U2AF65 as part of the core spliceosome machinery was used to exemplify the di↵erent processing steps and its known binding properties were recaptured (Zarnack et al, 2013). Next, I characterized the so far unknown binding spectrum of SRSF6 in human pancreatic beta-cells (Alvelos et al, 2020). The identified binding motif matched a recent description in mouse cells (Müller-McNicoll et al, 2016).…”

Section: Discussionmentioning

confidence: 89%

“…Technically such approaches make use of antisense oligonucleotides (ASOs) to block specific binding sites. As a proof of principle, we already used ASOs in the related publication to modulate the splicing of the LMO7 gene (Alvelos et al, 2020). In the case of SRSF6 binding sites on further susceptibility genes could be used as potential targets.…”

Section: Discussionmentioning

confidence: 99%

“…The project was carried out in cooperation with the group of Décio Eizirik and the group of Julian König (IMB, Mainz) and was recently published in the Life Science Alliance journal (Alvelos et al, 2020). The initial iCLIP experiment as well as protocol adaptation and optimization were performed by Ines Alvelos of the Eizirik group.…”

Section: The Role Of Srsf6 Binding In Human Pancreatic Beta-cellsmentioning

confidence: 99%

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Computational studies on RNA processing in higher eukaryotes

Brüggemann¹

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Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets. First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery. In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes. In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.

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

Cited by 17 publications

References 120 publications

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs

From Pancreatic β-Cell Gene Networks to Novel Therapies for Type 1 Diabetes

Computational studies on RNA processing in higher eukaryotes

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