An Exonic Splicing Enhancer in Human IGF-I Pre-mRNA Mediates Recognition of Alternative Exon 5 by the Serine-Arginine Protein Splicing Factor-2/ Alternative Splicing Factor
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Abstract:
The human IGF-I gene has six exons, four of which are alternatively spliced.Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment.To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron.Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%.We show that the serinearginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancerspecific manner.Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.Keywords:
Exonic splicing enhancer
Minigene
Splicing factor
SR protein
Spliceosome
Polypyrimidine tract
Protein splicing
Exon skipping
The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.
Spliceosome
Polypyrimidine tract
SR protein
Exonic splicing enhancer
Splicing factor
Protein splicing
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Two distinct functions have been proposed for the serine–arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein–protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.
Exonic splicing enhancer
SR protein
Spliceosome
Splicing factor
Protein splicing
Polypyrimidine tract
Minigene
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Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.
Minigene
Splicing factor
SR protein
Exonic splicing enhancer
Protein splicing
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Citations (34)
Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.
Polypyrimidine tract
Exonic splicing enhancer
Polypyrimidine tract-binding protein
SR protein
Protein splicing
Minigene
Splicing factor
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Citations (57)
SR proteins regulate alternative splicing by binding to exonic sequences where, via an arginine/serine-rich splicing activation domain, they enhance the binding of the spliceosome to the adjacent splice sites. Here, a system is described in which a nontoxic derivative of the small molecule rapamycin is used to control pre-mRNA splicing in vitro. This involves the rapamycin-dependent recruitment of a splicing activation domain located on one protein to a second protein bound to the pre-mRNA. These results provide a new approach to explore for regulating gene expression in vivo with small molecules by controlling pre-mRNA splicing.
SR protein
Spliceosome
Exonic splicing enhancer
Protein splicing
Polypyrimidine tract
Splicing factor
Minigene
Precursor mRNA
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Citations (14)
SR proteins play critical roles in the major pre-mRNA splicing pathway. A second pathway processes U12-dependent AT-AC introns. We demonstrate, by biochemical complementation, the requirement for SR proteins in splicing of AT-AC introns. Whereas SR proteins were sufficient to activate splicing of a P120 AT-AC intron, splicing of a sodium channel AT-AC intron required an additional nuclear fraction. Individual recombinant SR proteins promoted splicing of both substrates, but displayed marked preferences. SR proteins supported basal AT-AC splicing, and also splicing stimulation via a downstream enhancer or conventional 5' splice site. Analysis of chimeric transcripts revealed that information dispersed throughout exons and introns dictates SR protein specificity and the requirement for the additional nuclear fraction. Thus, SR proteins function in both major and minor splicing pathways, and in coordinating the activities of both spliceosomes via exon definition. These results suggest that despite the substantial differences in intron consensus sequences and in four of the five snRNPs in each spliceosome, at least some of the interactions involving SR proteins are conserved between the two pathways.
Spliceosome
SR protein
Exonic splicing enhancer
Protein splicing
Minor spliceosome
Splicing factor
snRNP
Polypyrimidine tract
Group II intron
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Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3′ untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3′ splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.
Minigene
SR protein
Exonic splicing enhancer
Protein splicing
Splicing factor
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Citations (43)
Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor
Julie L.C. Kan and Michael R. Green Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605 USA
Minigene
Exonic splicing enhancer
Polypyrimidine tract
Splicing factor
snRNP
SR protein
Protein splicing
Splice site mutation
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Citations (156)
The human IGF-I gene has six exons, four of which are alternatively spliced. Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment. To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron. Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%. We show that the serine-arginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancer-specific manner. Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.
Exonic splicing enhancer
Minigene
Splicing factor
SR protein
Polypyrimidine tract
Protein splicing
Spliceosome
Exon skipping
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Citations (46)
We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.
Minigene
Polypyrimidine tract
Exonic splicing enhancer
Splicing factor
SR protein
Splice site mutation
Protein splicing
Precursor mRNA
Exon skipping
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Citations (170)