Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.     

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.     

Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)₁₆ tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.     

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.     

Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes

Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.     

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.     

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.     

Direct repression of splicing by transformer-2

The Drosophila melanogaster sex determination factor Tra2 positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs but negatively affects the splicing of the M1 intron in tra2 pre-mRNA. Retention of the M1 intron is known to be part of a negative-feedback mechanism wherein the Tra2 protein limits its own synthesis, but the mechanism responsible for accumulation of M1-containing RNA is unknown. Here we show that the recombinant Tra2 protein specifically represses M1 splicing in Drosophila nuclear extracts. We find that the Tra2 protein binds directly to several sites in and near the M1 intron and that, when Tra2 binding is competed with other RNAs, the splicing of M1 is restored. Mapping the RNA sequences functionally required for M1 repression identified both a 34-nucleotide (nt) A/C-rich sequence immediately upstream of the M1 5' splice site and a region within the intron itself. The AC-rich sequence is largely composed of a repeated 4-nt sequence that also forms a subrepeat within the repeated 13-nt splicing enhancer elements of fru and dsx RNAs. Although required for repression, the element also enhances M1 splicing in the absence of Tra2. We propose that Tra2 represses M1 splicing by interacting with multiple sequences in the pre-mRNA and interfering with enhancer function.     

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.