Adenovirus mutants with splice-enhancing mutations in the E3 complex transcription unit are also defective in E3A RNA 3'-end formation

Region E3 of adenovirus encodes about 10 overlapping mRNAs with different spliced structures. The mRNAs are 5' coterminal and form two major 3'-coterminal families termed E3A and E3B. As a group, the mRNAs have two 5' splice sites and four or five 3' splice sites. We previously described a novel class of virus mutants with deletions that enhance distant upstream and downstream 5' and 3' splice sites in region E3 (S. L. Deutscher, B. M. Bhat, M. H. Pursley, C. Cladaras, and W. S. M. Wold, Nucleic Acids Res. 13:5771-5788, 1985). We now report that two of these mutants, dl710 and dl712, are defective in RNA 3'-end formation at the E3A site. This result was surprising because the deletions in dl710 and dl712 are upstream of the putative signal for E3A RNA 3'-end formation. The explanation that we favor for this result is that the enhanced splicing activity in these mutants results in the splicing out of the E3A 3'-end site from the RNA precursor before the E3A 3' ends have a chance to form.     

Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus

Region E3 of the adenovirus encodes about ten overlapping mRNAs (a to j) with different splicing patterns and with two RNA 3' end sites termed E3A and E3B. We have examined how deletions in 12 viable virus mutants affect differential RNA processing in E3. We assayed E3 mRNAs by the nuclease-gel and RNA blot procedures. Some deletions had no effect whereas others (e.g. deletion of a 3' splice or the E3A 3' end signal) had the anticipated effects on RNA processing. However, deletions in two regions had surprising effects. Deletions in one region (nucleotides 1691 to 2044) enhanced splicing at the upstream 951 5' splice site and the downstream 2157 and/or 2880 3' splice sites. Some of these deletions prevented RNA 3' end formation at the downstream E3A site. Deletion in the other region (nucleotides 2173 to 2237) enhanced an upstream splice site (951 to 2157) such that almost all pre-mRNA was processed into mRNA f. We suggest that these two regions contain cis-acting signals that regulate differential RNA processing. We discuss the results in terms of RNA folding and scanning models for splicing, as well as models for differential RNA 3' end formation at the E3A versus the E3B site.     

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.     

Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation.

The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.     

Optimization of a weak 3' splice site counteracts the function of a bovine papillomavirus type 1 exonic splicing suppressor in vitro and in vivo

Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3' splice site utilization from an early 3' splice site at nucleotide (nt) 3225 to a late-specific 3' splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3' splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3' splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3' splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3' splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3' splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3' splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3' splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3' splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3' splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3' splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.     

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.     

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.     

A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3' splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3' splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3' splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3' splice site at position 5639 in the late region. Optimization of the position 3358 3' splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3' splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.     

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.     

The cap and the 3' splice site similarly affect polyadenylation efficiency.

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.     

Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators.

We described previously the purification of a human protein, called alternative splicing factor (ASF), that can switch utilization of alternative 5' splice sites in an SV40 early pre-mRNA. We now report the isolation of a cDNA, designated ASF-1, that encodes this protein. ASF-1 consists of 248 amino acid residues, including an 80 residue RNA-binding domain at its N-terminus and a 50 residue C-terminal region that is 80% serine plus arginine. ASF-1 produced in E. coli can activate splicing in vitro and switch 5' splice-site utilization, establishing that the recombinant protein is sufficient to supply these activities. Analysis of additional cDNAs revealed that ASF pre-mRNA can itself be alternatively spliced, surprisingly, by utilization of a shared 5' splice site and two closely spaced 3' splice sites. Use of the upstream site results in a second mRNA (ASF-2) in which translation of the downstream exon occurs extensively in an alternative reading frame distinct from ASF-1.     

Selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor.

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. However, one 3' splice site, located at nucleotide (nt) 3225, is used for the processing of most BPV-1 pre-mRNAs in BPV-1-transformed C127 cells and at early to intermediate times in productively infected warts. At late stages of the viral life cycle, an alternative 3' splice site at nt 3605 is used for the processing of the late pre-mRNA. In this study, we used in vitro splicing in HeLa cell nuclear extracts to identify cis elements which regulate BPV-1 3' splice site selection. Two purine-rich exonic splicing enhancers were identified downstream of nt 3225. These sequences, designated SE1 (nt 3256 to 3305) and SE2 (nt 3477 to 3526), were shown to strongly stimulate the splicing of a chimeric Drosophila doublesex pre-mRNA, which contains a weak 3' splice site. A BPV-1 late pre-mRNA containing the nt 3225 3' splice site but lacking both SE1 and SE2 was spliced poorly, indicating that this 3' splice site is inherently weak. Analysis of the 3' splice site suggested that this feature is due to both a nonconsensus branch point sequence and a suboptimal polypyrimidine tract. Addition of SE1 to the late pre-mRNA dramatically stimulated splicing, indicating that SE1 also functions as an exonic splicing enhancer in its normal context. However, a late pre-mRNA containing both SE1 and SE2 as well as the sequence in between was spliced inefficiently. Further mapping studies demonstrated that a 48-nt pyrimidine-rich region immediately downstream of SE1 was responsible for this suppression of splicing. Thus, these data suggest that selection of the BPV-1 nt 3225 3' splice site is regulated by both positive and negative exonic sequences.