Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2

Abstract

The precise regulation of many alternative splicing (AS) events by specific splicing factors is essential to determine tissue types and developmental stages. However, the molecular basis of tissue-specific AS regulation and the properties of splicing regulatory networks (SRNs) are poorly understood. Here we comprehensively predict the targets of the brain-and muscle-specific splicing factor Fox-1 (A2BP1) and its paralog Fox-2 (RBM9) and systematically define the corresponding SRNs genome-wide. Fox-1/2 are conserved from worm to human, and specifically recognize the RNA element UGCAUG. We integrate Fox-1/2-binding specificity with phylogenetic conservation, splicing microarray data, and additional computational and experimental characterization. We predict thousands of Fox-1/2 targets with conserved binding sites, at a false discovery rate (FDR) of ∼24%, including many validated experimentally, suggesting a surprisingly extensive SRN. The preferred position of the binding sites differs according to AS pattern, and determines either activation or repression of exon recognition by Fox-1/2. Many predicted targets are important for neuromuscular functions, and have been implicated in several genetic diseases. We also identified instances of binding site creation or loss in different vertebrate lineages and human populations, which likely reflect fine-tuning of gene expression regulation during evolution.[Keywords: Tissue-specific alternative splicing; splicing regulatory network; Fox-1/A2BP1; Fox-2/RBM9; UGCAUG; comparative genomics] Supplemental material is available at http://www.genesdev.org.Received June 6, 1007; revised version accepted July 28, 2008.The sequencing of complete genomes revealed that complex metazoans, including mammals, have only slightly more genes than unicellular yeast (International Human Genome Sequencing Consortium 2001). Organismal complexity must have resulted largely from mechanisms for diversifying the expression products, and the temporal and spatial patterns, from a limited set of genes. It is crucial to understand how gene expression is orchestrated to determine developmental stages, specify cell types, and respond to external stimuli (Maniatis and Reed 2002). Alternative splicing (AS), the process for removing introns from pre-mRNA transcripts and joining exons in different combinations, is an essential step of post-transcriptional regulation (Cartegni et al. 2002;Black 2003). In mammals, more than two-thirds of genes are alternatively spliced (Johnson et al. 2003). The choice of exons and splice sites is largely determined by many RNA-binding proteins, or splicing factors, which interact with cis-regulatory elements to activate or repress particular splicing events.Many splicing factors have restricted and dynamic expression patterns, and play important roles in tissue-specific or developmentally regulated splicing of particular transcripts. However, the mechanisms and impact of these AS events remain poorly understood. A well-studied example is Sxl, Tra, Tra-2, and several other s...

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

“…We again used the MS2-tethering assay to examine RBFOX1's activity in exon repression. We modified a b-globin PB1 minigene (Zhang et al 2008) to address this question. We mutated the three natural RBFOX1 motifs in the upstream intron, as well as two motifs in the exon.…”

“…Global analysis was utilized more recently to evaluate the splicing regulatory networks of the RBFOX family of proteins. A combination of microarray, CLIP-seq, high-throughput RT-PCR, and computational analyses by different laboratories resulted in the identification of many endogenous target genes, and confirmed the so-called ''RNA map,'' meaning that the splicing outcome (activation or repression) depends in a predictable manner on the location of the UGCAUG element (Zhang et al 2008;Venables et al 2009;Yeo et al 2009). Certain other splicing factors, such as Nova and PTB, also have a similar map with position-dependent activities (Ule et al 2006;Llorian et al 2010).…”

“…They usually promote exon inclusion when binding to the intron downstream from an alternative cassette exon, and exon skipping when binding to the upstream intron (Jin et al 2003;Underwood et al 2005;Ponthier et al 2006;Zhou et al 2007;Zhang et al 2008;Tang et al 2009;Yeo et al 2009). Several target genes have been extensively studied by use of reporter minigenes such as mitochondrial ATP synthase 3 g-subunit (F1g) (Jin et al 2003), calcitonin/calcitoningene-related peptide (CGRP) (Zhou et al 2007), CaV1.2 L-Type calcium channel ), fibronectin (Jin et al 2003), non-muscle myosin II heavy chain-B (NMHC-B) (Nakahata and Kawamoto 2005), epithelial cell-specific fibroblast growth factor receptor 2 (FGFR2) (Baraniak et al 2006), and RBFOX1 and RBFOX2 themselves (Damianov and Black 2010).…”

Functional correction by antisense therapy of a splicing mutation in the GALT gene

“…31,32 Altogether, in vivo and ex vivo analyses demonstrated that the c.820 þ 13A4G variation is actually a disease-causing mutation. Our results, neatly complemented by the in vivo analysis of patients' cells, reiterate the reliability of the ex vivo minigene functional assay to unravel the molecular mechanism(s) whereby a variation affects splicing.…”

RNA-binding proteins in neurological diseases

“…Interestingly, many of the predicted Rbfox2 alternatively spliced target transcripts themselves encoded splicing factors, suggesting the possibility of a higher-order regulatory network. Gene ontology (GO) analysis of the Rbfox2 splicing regulatory network demonstrated enrichment of a biologically coherent set of transcripts encoding proteins mediating synaptic transmission and membrane excitability [77,78]. Genome-wide analysis of brain RNA of Rbfox2 knock-out mice identified numerous splicing changes altering proteins important both for brain development and mature neuronal function.…”