Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition

Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

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.     

U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression

Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5' splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5' splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and?an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.     

Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.     

Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity.

A functional analysis of exon replacement mutations was performed in parallel with RNA-protein binding assays to gain insight into the role of the exon in alternative and simple splicing events. These results show that constitutive exons from unrelated genes contain strong signals that promote splicing in multiple sequence contexts by enhancing 3' splice site activity. A clue to the nature of the relationship between the exon and adjacent 3' splice site is indicated by the binding properties of exon variant RNAs when tested with different biochemical preparations of the essential splicing protein, U2AF. In the context of a complete nuclear extract, U2AF binding to the 3' splice site is stimulated by the presence of an adjacent constitutive exon. In contrast, highly purified HeLa U2AF binds equivalently to the exon variants under conditions in which differential polypyrimidine tract binding is evident. These results provide support for an assisted binding model in which positive-acting signals within exons, exon enhancers, direct the binding of accessory factors, which in turn increase the intrinsic affinity of U2AF for the adjacent 3' splice site. Further support for an assisted binding model is indicated by biochemical complementation of U2AF binding and by the localization of a novel exon enhancer, which, when introduced into a weak exon, stimulates splicing activity in parallel with U2AF binding. Immunoprecipitation analysis identifies the splicing factor, SC35, as a constituent of the exon enhancer binding complex. These results are discussed in the context of current models for functional exon-bridging interactions.     

Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.     

Repression of alpha-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract

Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the alpha-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the alpha-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 3' end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF(65) binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF(65), thereby specifically blocking splicing of the SM exon.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.     

The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.     

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.     

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.     

Polypyrimidine track-binding protein binding downstream of caspase-2 alternative exon 9 represses its inclusion

We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3' portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3' acceptor site that engages in nonproductive commitment complex interactions with the 5' splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3' acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3' splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism.