Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins.

In addition to the protein products of the adenovirus E1A 13S and 12S mRNAs, monoclonal antibodies specific for the E1A proteins immunoprecipitate polypeptides with relative mol. wt of 30,000 (30 kd) and 35,000 (35 kd) from extracts of infected cells. The 30 kd and 35 kd proteins are encoded by novel mRNAs referred to as the 10S and 11S mRNAs, respectively. These two mRNAs arise from differential splicing of the E1A precursor RNA. For the 10S mRNA, the precursor is spliced twice, once removing the region between nucleotides 637 and 854 and once between 974 and 1229. The splice between nucleotides 974 and 1229 is identical to the one used for the processing of the 12S mRNA. Synthesis of the 11S mRNA also utilizes two splicing events. One of these is identical to the 637/854 splice of the 10S mRNA, and the other removes the region between nucleotides 1112 and 1229, a splice junction also found in the 13S mRNA. All four mRNAs used the same reading frame and, therefore, code for related proteins. The products of the 10S and 11S mRNAs are identical to the products of the 12S and 13S mRNAs, respectively, except for an internal stretch of 27 amino acids removed by the 637/854 splice. Within this segment is a group of amino acid residues that is highly conserved between different adenovirus serotypes. Mutant adenoviruses in which the wild-type E1A sequences have been replaced with cDNA copies of the 10S or 11S mRNAs are defective for growth on HeLa cells suggesting that this region is important for viral growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of intron length on differential processing of mouse mu heavy-chain mRNA.

Production of membrane-bound and secreted forms of mouse mu heavy-chain mRNA is controlled by differential processing in a developmental-stage-specific manner. We have analyzed the effects of various deletions and insertions in the C4-M1 intron of the mouse mu gene on the differential processing of mu mRNA. We show that there is a correlation between the length of the C4-M1 intron and the molar ratio of membrane-bound to secreted mu mRNAs, i.e., the shorter the C4-M1 intron, the higher the ratio. Since the poly(A) addition signal in the C4-M1 intron seems to be intact in the mutant mu genes, it is likely that the efficiency of splicing of the C4-M1 intron is affected by changes in the intron length.     

Nuclei of adenovirus 2-infected cells contain an RNA species that corresponds to an intron excised intact from mRNA precursors.

We used Northern and S1 nuclease analyses to identify a nuclear RNA species of the structure predicted for an intron excised from the precursor of adenovirus 2 E2A early mRNA. The structure of this excised intron demonstrated that the splicing of E2A mRNA can involve the removal of the intron sequences as single intact molecules. The concentration of the excised intron is 30 copies per nuclei, comparable to the levels of E2A polyadenylated splicing precursors, but 30-fold less than nuclear E2A mRNA. Late in infection, when the production of E2A early mRNA is dramatically elevated (Goldenberg et al., J. Virol. 38:932-939, 1981), the excess intron was not detected, indicating that either its stability or the mechanism of splicing is altered. We also identified a spliced nonpolyadenylated E2A nuclear RNA species with a structure similar to the mRNA, indicating that splicing may normally occur in the absence of polyadenylation.     

A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.

We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel splicing of the E1A mRNA precursor, minor changes in the ratio of E1A 12 to 13S mRNAs, and modest nuclear accumulation of beta-globin mRNA precursors with either one of the two introns removed. Most of the altered U1 snRNPs did not affect the level of mature cytoplasmic mRNA significantly, but in one case an altered U1 snRNP (alpha 1) whose intended target was located downstream from the adenovirus E1A 12S 5' splice site was able to reduce the level of cytoplasmic 12S mRNA by approximately 60% and that of 13S mRNA by 90%. This alpha 1 snRNP induced an additional E1A splice, resulting in the appearance of 10 and 11S E1A mRNAs normally found only late in adenovirus infection. Thus, a trans-acting factor can induce alternative splicing. Surprisingly, the effects of alpha 1 on E1A splicing were not abolished by deleting the intended target sequence on the mRNA precursor.     

Relationship between mRNA stability and intron presence

Introns were found to enhance almost every steps of gene expression except increasing mRNA stability. By analyzing the genome-wide data of mRNA stability published by someone previously, we found that human intron-containing genes have more stable mRNAs than intronless genes, and the Arabidopsis thaliana genes with the most unstable mRNAs have fewer introns than other genes in the genome. After controlling for mRNA length, we found mRNA stability is still positively correlated with intron number in human intron-containing genes. But in yeast Saccharomyces cerevisiae, two different datasets on mRNA half-life gave conflicting results. The components of messenger ribonucleoprotein particles recruited during intron splicing may be retained in cytoplasmic mRNPs and act as signals of mRNA stability or simply insulators to avoid mRNA degradation.     

Splicing of the E2A premessenger RNA of adenovirus serotype 2. Multiple pathways in spite of excision of the entire large intron

During the early period of infection, the 4.9 kb (kb = 10(3) bases) E2A premRNA of adenovirus serotype 2 is matured mainly into a 2.0 kb mRNA by excision of introns of 2233 and 626 nucleotides. In order to define all the possible steps of splicing occurring in vivo, we characterized splicing intermediates present after a limiting treatment of cells with cycloheximide. Three complementary methods of analysis were used: RNA transfer analysis, S1 nuclease mapping and complementary DNA-RNase assay. Our principal conclusions concerning the poly(A)+ species are as follows. The RNA intermediate family detected is more complex than expected, since two major RNA intermediates of 4.6 kb and 4.3 kb, two minor intermediates of 2.9 kb and 2.6 kb, and a 2.3 kb RNA, which represents a minor alternative mRNA form, are revealed. Despite its large size and the presence of multiple internal donor and acceptor signals, intron 1 is exclusively excised as a whole. Intron 2 is either primarily excised as a whole, removing the standard 626-nucleotide sequence, or a smaller sequence of 337 nucleotides is removed, generating the 2.3 kb alternative mRNA. Kinetics of the ligation reaction demonstrate that the minimal time for excision of intron 2 is no more than two minutes, indicating a high level of co-ordination of the multiple individual reactions occurring during excision of an intron. Besides the major pathway for E2A premRNA splicing, namely the excision first of intron 2, followed by the excision of intron 1 after a lag time of five minutes, a minor pathway (used with a frequency of 10%) can be detected where the order of intron excision was inverted. With the alternative variant of excision of intron 2, at least three different pathways are therefore used to mature the E2A premRNA. RNA intermediates resulting from the cleavage at the 5' end of introns and branching can be detected by S1 mapping experiments, but their low accumulative level (1% relatively to the initial premRNA) precluded their direction by RNA transfer experiments and their complete characterization.     

The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function

We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.     

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.     

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.