Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.     

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing

We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.     

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.     

Amplification of polyomavirus DNA sequences stably integrated in rat cells.

To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous sequences.     

Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites

To explore the mechanism and control of alternative splicing, we have characterized the products formed by splicing of SV40 early pre-mRNA in vitro and in vivo. Large T and small t mRNAs are derived from this precursor by joining alternative 5' splice sites to a single shared 3' splice site. In contrast to pre-mRNAs studied previously, we have shown that splicing to large T RNA involves the utilization of multiple lariat branch sites, while small t splicing uses a single branch site. Interestingly, the predominant branch sites utilized in splicing of large T RNA in vitro were found to differ in nuclear extracts from HeLa and human 293 cells, correlated with previously observed differences in the ratio of large T to small t mRNAs produced in the two cell types. To test the significance of this correlation, we examined the products formed by splicing of an SV40 early precursor microinjected into X. laevis oocytes. Strikingly, both the pattern of branch sites used in large T splicing and the ratio of large T to small t mRNAs produced were found to be identical to those observed in 293 cells and extracts.     

Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.     

The role of the polypyrimidine stretch at the SV40 early pre-mRNA 3'' splice site in alternative splicing.

We have studied the role in pre-mRNA splicing of the nucleotide sequence preceding the SV40 early region 3' splice site. Somewhat surprisingly, neither the pyrimidine at the highly conserved -3 position, nor the polypyrimidine stretch that extends from -5 to -15, relative to the 3' splice site, were found to be required for efficient splicing. Mutations that delete this region or create polypurine insertions at position -2 had no significant effects on the efficiency of SV40 early pre-mRNA splicing in vivo or in vitro. Interestingly, however, the pyrimidine content of this region had substantial effects on the alternative splicing pattern of this pre-mRNA in vivo. Mutations that increased the number of pyrimidine residues resulted in more efficient utilization of the large T antigen mRNA 5' splice site relative to the small t 5' splice site, while mutations that increased the purine content enhanced small t mRNA splicing. A possible molecular mechanism for these findings, as well as a model that proposes a role for the polypyrimidine stretch in alternative splicing, are discussed.     

Identification of an hnRNP A1-dependent splicing silencer in the human papillomavirus type 16 L1 coding region that prevents premature expression of the late L1 gene

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3' splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3' splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5' splice site in the early region to the L1 3' splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.     

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.     

Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii.

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.     

Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains

In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.     

Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells.

The great majority of viral mRNAs in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus signal. Twenty percent of BPV mRNA from productively infected cells bypasses the early polyadenylation site and uses the late polyadenylation site approximately 3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this mutation did not result in significant read-through expression of downstream RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites approximately 100 nt upstream of the wild-type early polyadenylation site. In addition, cells transformed by wild-type BPV harbor a small population of mRNAs with 3' ends located in this upstream region. These experiments demonstrate that inactivation of the major poly(A) signal induces preferential use of otherwise very minor upstream poly(A) sites. Mutational analysis suggests that polyadenylation at the minor sites is controlled, at least in part, by UAUAUA, an unusual variant of the poly(A) consensus signal approximately 25 nt upstream of the minor polyadenylation sites. These experiments indicate that inactivation of the major early polyadenylation signal is not sufficient to induce expression of the BPV late genes in transformed mouse cells.