The mammalian homolog of suppressor-of-white-apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS

We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(wa) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5' splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5' splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire IIICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors.     

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.     

Identification of exon sequences involved in splice site selection

The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons. When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct. Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals. A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold. Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold. Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection.     

Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Regulation of alternative polyadenylation by U1 snRNPs and SRp20

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3'-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3'-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3'-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3' and 5' splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3'-terminal exons and perhaps in the communication between polyadenylation and splicing.     

Interaction between the negative regulator of splicing element and a 3' splice site: requirement for U1 small nuclear ribonucleoprotein and the 3' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.     

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.     

An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.     

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

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.     

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator

Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.     

Bimolecular exon ligation by the human spliceosome

Intron excision is an essential step in eukaryotic gene expression, but the molecular mechanisms by which the spliceosome accurately identifies splice sites in nuclear precursors to messenger RNAs (pre-mRNAs) are not well understood. A bimolecular assay for the second step of splicing has now revealed that exon ligation by the human spliceosome does not require covalent attachment of a 3' splice site to the branch site. Furthermore, accurate definition of the 3' splice site in this system is independent of either a covalently attached polypyrimidine tract or specific 3' exon sequences. Rather, in this system 3' splice site selection apparently occurs with a 5' --> 3' directionality.     

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.     

Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.     

CA- and purine-rich elements form a novel bipartite exon enhancer which governs inclusion of the minute virus of mice NS2-specific exon in both singly and doubly spliced mRNAs

The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5'-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5' and 3' regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3' splice site upstream (i.e., the exon 5' terminus) or a strong 5' splice site downstream (i.e., the exon 3' terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3' splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3' splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5' splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.