Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16 PDF Download
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Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.
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Languages : en
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Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.
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A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.
Author: Mohini Rohit Jangi
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Languages : en
Pages : 216
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Regulated alternative splicing is mediated by RNA binding proteins (RBPs) recognizing short sequence motifs in nascent transcripts. The Rbfox RBPs are highly conserved splicing factors that regulate tissue-specific exon inclusion by binding the RNA motif UGCAUG. We sought to comprehensively define the Rbfox2 splicing regulatory network and discover determinants of Rbfox2 regulation in mouse embryonic stem cells. We uncovered fundamental principles in the mechanistic aspects of Rbfox-dependent splicing and in the systems-level regulation of interconnected splicing networks. Using high-resolution iCLIP and RNAseq, we identified many Rbfox2-mediated proteincoding splicing events and nearly 300 additional events, in particular those within RBPs, that are coupled to nonsense-mediated mRNA decay (NMD). Regulation of NMD-coupled splicing by Rbfox2 alters gene expression of autoregulated RBPs and hundreds of additional genes. These observations place Rbfox2 upstream of a large network of direct and indirect splicing changes and offer an explanation as to how autoregulated gene expression can be modulated. We describe a validation of RNA Bind-n-Seq, a novel in vitro technique for analyzing RNA-protein interactions. We found a secondary Rbfox2 motif, GCACG, to be functional in splicing regulation in addition to the consensus UGCAUG and observed a preference for Rbfox2 binding to unstructured sequences. These findings provide a foundation for establishing the critical determinants of functional cis elements in splicing regulation. We also investigated mechanisms of co-transcriptional splicing regulation by Rbfox2. Using chromatin immunoprecipitation, we found that Rbfox2 is recruited early in the transcription cycle to active promoters and transcriptional enhancers, likely via interaction and co-transcriptional tracking with RNA polymerase II. Modulation of chromatin structure alters Rbfox2-dependent splicing activity, supporting the emerging model that the chromatin environment influences exon choice. Our analyses of Rbfox2 activity in mouse embryonic stem cells reveal hundreds of previously unknown splicing targets involved in diverse biological functions. In particular, we introduce a novel concept in splicing regulation whereby changes in the expression of one splicing factor induce a cascade of secondary and perhaps tertiary splicing changes through cross-regulation of RBPs. This model positions RBPs at a critical node in the establishment, reinforcement, and alteration of tissue transcriptomes during mammalian development.
Author: Sandya Ajith
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Languages : en
Pages : 182
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Alternative splicing is the process by which an exon is preferentially included or excluded from an mRNA transcript. Recent global sequencing studies have shown that >95% of the transcriptome undergoes some form of alternative splicing. Such regulation often alters protein isoform expression, as is especially apparent in T cells of the immune system that change their expression of RNA and protein according to signaling cues. The focus of this thesis is on one alternative exon in the pre-mRNA of transcription factor LEF1 and its regulation by the splicing factor CELF2. LEF1 is crucial for T cell function as it upregulates the expression of TCR[alpha]. Upon signal induction in T-cells, CELF2 promotes the inclusion of exon 6 in LEF1 (LEF1-E6) in the final mRNA transcript. This increase in LEF-E6 inclusion generates an isoform of LEF1 that is preferentially active in promoting transcription of TCR[alpha]. CELF2 regulates LEF1-E6 inclusion upon stimulation by increasing its binding to two conserved elements (USE60 and DSE120) in the upstream and downstream introns flanking exon 6. My goal is to understand how the increase of binding of CELF2 to the USE60 and DSE120 upon stimulation results in an increase in LEF1-E6 inclusion. Using a combination of in vivo minigene assays, in vitro splicing assays and UV-crosslinking assays I correlate the binding of CELF2 to the function of the USE60 and DSE120. I show that the USE60 and DSE120 do not work synergistically to enhance inclusion but function antagonistic to each other. The USE60 is a repressor of splicing while the DSE120 is an enhancer. In order to achieve an increase in exon 6 inclusion only upon stimulation, CELF2 binding is highly regulated between the USE60 and DSE120. In unstimulated T cells, binding is biased towards the repressive USE60 and upon stimulation the increase in CELF2 binding happens purely on the activating DSE120. This bolus of CELF2 binding on the DSE120 upon stimulation leads to an increase in exon 6 inclusion. These studies reveal a model where binding of CELF2 to the DSE120 is inhibited in unstimulated cells and this inhibition is relieved upon stimulation.
