Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.     

Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins.

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.     

ESEfinder: A web resource to identify exonic splicing enhancers

Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.     

Regulation of SRp20 exon 4 splicing

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.     

Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer

The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5′ splice site (ss) or 3′ ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3′ ss selectivity within the 3′ ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5′ ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3′ ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.     

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.     

General and specific functions of exonic splicing silencers in splicing control

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.     

Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.     

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy

SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.     

SR proteins Asf/SF2 and 9G8 interact to activate enhancer-dependent intron D splicing of bovine growth hormone pre-mRNA in vitro

The alternative splicing of the last intron (intron D) of bovine growth hormone (bGH) pre-mRNA requires a down-stream exonic splicing enhancer (FP/ESE). The presence of at least one SR protein has been shown to be essential for FP/ESE function and splicing of intron D in in vitro splicing assays. However, in vitro reconstitution of splicing using individual purified SR proteins may not accurately reflect the true complexity of alternative splicing in an intact nucleus, where multiple SR proteins in varying amounts are likely to be available simultaneously. Here, a panel of recombinant baculovirus-expressed SR proteins was produced and tested for the ability to activate FP/ESE-dependent splicing. Individual recombinant SR proteins differed significantly in their activity in promoting intron D splicing. Among the recombinant SR proteins tested, SRp55 was the most active, SC35 showed very little activity, and ASF/SF2 and 9G8 individually had intermediate activity. At least one SR protein (ASF/SF2) bound to the FP/ESE with characteristics of a cooperative interaction. Most interestingly, low concentrations of ASF/SF2 and 9G8 acted synergistically to activate intron D splicing. This was due in part to synergistic binding to the FP/ESE. Splicing of bGH intron D is inherently complex, and is likely controlled by an interaction of the FP/ESE with several trans-acting protein factors acting both independently and cooperatively. This level of complexity may be required for precise control of alternative splicing by an exon sequence, which simultaneously is constrained to maintain translational integrity of the mature mRNA.     

Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5' splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5' splice site. Second, utilization of the weak M2 5' splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine-rich splicing enhancer sequence that is located in the 3' exon of M1 mRNA. We demonstrate that activation of the M2 5' splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.     

Exonic splicing enhancer motif recognized by human SC35 under splicing conditions

Exonic splicing enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under splicing conditions. The selected sequences are functional and specific: they promote splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific enhancers in natural exon sequences.     

A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates human immunodeficiency virus type 1 rev, env, vpu, and nef gene expression

The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.