Tissue-Specific Splicing

Abstract: Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue-specific RNA-binding protein, Fox-1, which regulates alternative splicing in vertebrates. Fox-1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox-1 is expressed in heart and skeletal muscles. As candidates for muscle-specific targets of Fox-1, we considered two genes, the human mitochondrial ATP synthase γ-subunit gene (F1γ) and the rat α-actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox-1 induced muscle-specific exon skipping of the F1γ gene via binding to GCAUG sequences upstream of the regulated exon. Fox-1 also regulated mutually exclusive splicing of the α-actinin gene, antagonizing the repressive effect of polypyrimidine tract-binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox-1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox-1 plays key roles in both positive and negative regulation of tissue-specific splicing via GCAUG.

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 approximately 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.

Abstract: The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific splicing decisions most likely result from differences in the concentration and/or activity of these proteins. However, large-scale data to systematically address this issue have just recently started to become available. Here we show that splicing factor gene expression signatures can be identified that reflect cell type and tissue-specific patterns of alternative splicing. We used a computational approach to analyze microarray-based gene expression profiles of splicing factors from mouse, chimpanzee and human tissues. Our results show that brain and testis, the two tissues with highest levels of alternative splicing events, have the largest number of splicing factor genes that are most highly differentially expressed. We further identified SR protein kinases and small nuclear ribonucleoprotein particle (snRNP) proteins among the splicing factor genes that are most highly differentially expressed in a particular tissue. These results indicate the power of generating signature-based predictions as an initial computational approach into a global view of tissue-specific alternative splicing regulation.

Abstract: The beta-tropomyosin gene in chicken contains two mutually exclusive exons (exons 6A and 6B) which are used by the splicing apparatus in myogenic cells, respectively, before (myoblast stage) and after (myotube stage) differentiation. The myoblast splicing pattern is shown to depend on multiple sequence elements that are located in the upstream intron and in the exon 6B and that exert a negative control over exon 6B splicing. This regulation of splicing is due, at least in part, to a secondary structure of the primary transcript, which limits in vivo the accessibility of exon 6B in myoblasts.