HITS-CLIP and Integrative Modeling Define the Rbfox Splicing-Regulatory Network Linked to Brain Development and Autism

Abstract

Summary The RNA-binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here we performed HITS-CLIP for all three Rbfox family members to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We found that the two guanines in the Rbfox-binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites combined with additional datasets defined 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with down-regulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing- regulatory network.

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

“…The similar phenotype observed for Rbfox1 and Rbfox2 KD suggests that both RBPs regulate overlapping sets of transcripts in beta cells. This is in agreement with previous studies showing that Rbfox proteins bind to the same UGCAUG motif due to the presence of a common RNA binding domain (28,32,62). Functional analysis of the role of Rbfox proteins in beta cells (present data) indicates that Rbfox depletion increases the insulin secretory capacity by affecting actin depolymerization kinetics following glucose stimulation.…”

“…Gelsolin enhances insulin secretion by mediating glucose-dependent actin cytoskeleton remodeling and by interacting with t-SNARE proteins, thus favoring insulin granule exocytosis (29,30). In line with the present findings, Rbfox1 targets in brain and muscle are enriched in genes involved in cytoskeleton organization and actin filament-based processes (28,57). We cannot exclude, however, that other Rbfox targets (such as calcium channels) also contribute to the observed phenotype.…”

“…Our data analysis identified a cluster of RBPs that are expressed in both brain and in human islets. This cluster includes members of the Elavl, Nova, and Rbfox families, which have been implicated in neuronal physiology and disease (28,(31)(32)(33)(34)(35). We found that Elavl4 and Nova2 are required for beta cell survival, and their depletion leads to activation of the intrinsic pathway of apoptosis.…”

“…Rbfox1 Regulates Alternative Splicing of Key Genes Controlling Insulin Secretion-To identify Rbfox target genes that might affect insulin release, we re-analyzed a previously published dataset that identified 1,059 Rbfox-regulated AS events in mouse brain (28). Pathway enrichment analysis using IPA (Ingenuity Systems) and DAVID (david.abcc.ncifcrf.gov) software showed that Rbfox-regulated transcripts in brain are enriched in pathways that can affect insulin secretion in beta cells, including cytoskeleton organization, vesicle-mediated transport, and calcium signaling.…”

Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

“…S12). Rbfox and Nova are known to regulate AS of neuronal transcripts by activating or repressing exon inclusion depending on their binding position relative to the alternative exon (17,20,21). We found that both known and new cassette exons with neuron-specific inclusion 3B and SI Appendix, Fig.…”