Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo.

The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.     

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.     

Yeast pre-mRNA splicing requires a minimum distance between the 5'' splice site and the internal branch acceptor site.

We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.     

Switch in 3' splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation

Maintenance of female sexual identity in Drosophila melanogaster involves an autoregulatory loop in which the protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. We have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in regulation. Our results indicate that exon 3 repression requires competition between the 5' splice sites of exons 2 and 3 but is independent of their relative strength. Two 3' splice site AG's precede exon 3. We report here that, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, arguing for a switch in 3' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3' splice sites is important for the efficient regulation by SXL, suggesting that SXL interferes with molecular events occurring between initial splice site communication across the exon and the splice site pairing that leads to intron removal.     

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.     

U5 snRNA interacts with exon sequences at 5' and 3' splice sites.

U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.     

Yeast mRNA splicing in vitro

Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo.     

Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast.

U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs. We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals. Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block. Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA. Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA. U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA.     

Activation of a cryptic 5' splice site by U1 snRNA

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.     

A general role for splicing enhancers in exon definition

Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.     

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3' splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3' splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3' splice sites.     

Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.     

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.     

Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3' splice site pairing

Spinal muscular atrophy is caused by the loss of functional survival motor neuron (SMN1) alleles. A translationally silent nucleotide transition in the duplicated copy of the gene (SMN2) leads to exon 7 skipping and expression of a nonfunctional gene product. It has been suggested that differential SMN2 splicing is caused by the disruption of an exonic splicing enhancer. Here we show that the single nucleotide difference reduces the intrinsic strength of the 3' splice site of exon 7 2-fold, whereas the strength of the 5' splice site of the exon 7 is not affected. Thus, a decrease in splice site strength is magnified in the context of competing exons. These data suggest that lower levels of exon 7 definition not only reduce intron 6 removal but, more importantly, increase the efficiency of the competing exon 7 skipping pathway. Antisense oligonucleotides were tested to modulate exon 7 inclusion, which contains the authentic translation stop codon. Oligonucleotides directed toward the 3' splice site of exon 8 were shown to alter SMN2 splicing in favor of exon 7 inclusion. These results suggest that antisense oligonucleotides could be used as a therapeutic strategy to counteract the progression of SMA.