Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

Lovci, Michael T.; Ghanem, Dana; Marr, Henry; Arnold, Justin D.; Gee, Sherry L.; Parra, Marilyn; Liang, Tiffany Y.; Stark, Thomas; Gehman, Lauren; Hoon, Shawn; Massirer, Katlin B.; Pratt, Gabriel A.; Black, Douglas L.; Gray, Joe W.; Conboy, John G.; Yeo, G

doi:10.1038/nsmb.2699

Cited by 338 publications

(387 citation statements)

References 70 publications

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“…Alternatively, this may be related to the observed Nova1 up-regulation upon Nova2 silencing, revealing a compensatory mechanism that could decrease physiological perturbations due to changes in splicing pathways. Reciprocal compensatory regulation between RBP paralogs has been previously observed in the Nova and other RBP families (49,50).…”

Section: Role Of Neuron-enriched Rna-binding Proteins In Beta Cellsmentioning

confidence: 94%

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Juan-Mateu

Rech

Villate

et al. 2017

Journal of Biological Chemistry

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Edited by Jeffrey E. PessinPancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-␤H1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.Insulin-secreting pancreatic beta cells share many phenotypic traits with neurons. Similarities range from an analogous physiology and function to similar development and gene expression (1). Beta cells release insulin using a similar exocytotic machinery as used by neurons to release neurotransmitters. Indeed, insulin is stored and secreted using scaffolding proteins and synaptic-like vesicles similar to neuronal cells (2-4), and like neurons, beta cells are able to generate action potentials in response to different stimuli (5). Despite having different embryonic origins (6, 7), global gene expression and active chromatin marks of beta cells are closer to neural tissues than to any other tissue, including other pancreatic cells (8). Neurons are phylogenetically older than beta cells, and in some primitive organisms neurons express insulin and regulate circulating glucose levels (9, 10). Taken together, these findings suggest that beta cells have evolved into specialized insulinsecretory cells by adopting, at least in part, neuronal transcription programs (1). Supporting this hypothesis, both neurons and beta cells lose the expression of the transcriptional repressor element-1 silencing transcription factor (REST) 5 during differentiation (11). REST is expressed in most non-neuronal cells and acts as a master negative regula...

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Section: Role Of Neuron-enriched Rna-binding Proteins In Beta Cellsmentioning

confidence: 94%

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Juan-Mateu

Rech

Villate

et al. 2017

Journal of Biological Chemistry

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“…3B). Interestingly, a subset of enhanced exons also contained UGCAUG motifs proximal to the downstream constitutive 39 splice site, perhaps implicating distant motifs in Rbfox2-dependent exon inclusion (Lim and Sharp 1998;Lovci et al 2013). Most of the motif occurrences near regulated exons were well conserved.…”

Section: Rbfox2 Rna Map Reveals Position-dependent Regulation Of Cassmentioning

confidence: 99%

“…Statistically significant genic clusters of FHFOX2 binding were determined next using cross-link sites extended on either side by 12 nt as input into the CLIPper algorithm (available at https://github.com/YeoLab/clipper) (Lovci et al 2013). Allowing for the calling of clusters within pre-mRNA regions, we identified 40,243 FHFOX2 clusters and only 2961 UntagFOX2 clusters (gene-level false discovery rate [FDR] # 0.05; P # 0.05).…”

Section: Rbfox2 Individual Nucleotide-resolution Clip (Iclip) Is Enrimentioning

confidence: 99%

“…Crosslink sites were determined from 59-terminal nucleotides of contiguously mapping reads and deletion sites of reads mapping with deletions. FHFOX2 cross-links extended by 12 nt on either side were clustered using the Clipper algorithm (available at https://github.com/YeoLab/clipper) (Lovci et al 2013). FHFOX2 clusters significantly enriched over UntagFOX2 read coverage were considered for further analysis.…”

Section: Iclip Mapping and Clusteringmentioning

confidence: 99%

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Rbfox2 controls autoregulation in RNA-binding protein networks

Boutz²,

et al. 2014

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The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsensemediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to highthroughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These ''silent'' events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMDinducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

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“…Recent studies on RSSs in eukaryotic genes revealed widespread occurrence of long-range RRI with diverse functions such as riboswitches (Li and Breaker 2013) and mediators of exon skipping (Lovci et al 2013), mutually exclusive exon choice (Kreahling and Graveley 2005;Yang et al 2011), and other types of alternative splicing events (Raker et al 2009;Pervouchine et al 2012). Many of these structures are located in regions lacking reliable sequence alignments and contain long, ultraconserved stretches of complementary nucleotides, in which the interacting bases can be separated by distances as large as 10 kb.…”

Section: Introductionmentioning

confidence: 99%

IRBIS: a systematic search for conserved complementarity

Pervouchine¹

2014

RNA

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IRBIS is a computational pipeline for detecting conserved complementary regions in unaligned orthologous sequences. Unlike other methods, it follows the "first-fold-then-align" principle in which all possible combinations of complementary k-mers are searched for simultaneous conservation. The novel trimming procedure reduces the size of the search space and improves the performance to the point where large-scale analyses of intra-and intermolecular RNA-RNA interactions become possible. In this article, I provide a rigorous description of the method, benchmarking on simulated and real data, and a set of stringent predictions of intramolecular RNA structure in placental mammals, drosophilids, and nematodes. I discuss two particular cases of long-range RNA structures that are likely to have a causal effect on single-and multiple-exon skipping, one in the mammalian gene Dystonin and the other in the insect gene Ca-α 1 D. In Dystonin, one of the two complementary boxes contains a binding site of Rbfox protein similar to one recently described in Enah gene. I also report that snoRNAs and long noncoding RNAs (lncRNAs) have a high capacity of base-pairing to introns of protein-coding genes, suggesting possible involvement of these transcripts in splicing regulation. I also find that conserved sequences that occur equally likely on both strands of DNA (e.g., transcription factor binding sites) contribute strongly to the false-discovery rate and, therefore, would confound every such analysis. IRBIS is an open-source software that is available at

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Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

Cited by 338 publications

References 70 publications

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Rbfox2 controls autoregulation in RNA-binding protein networks

IRBIS: a systematic search for conserved complementarity

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