Complex splicing patterns of RNAs from the early regions of adenovirus-2 (original) (raw)
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The spliced structures of adenovirus 2 fiber message and the other late mRNAs
Cell, 1978
All late adenovirus 2 messenger RNAs, except perhaps that for peptide IX, are composite molecules with sequences derived from at least two to five or more separate portions of the genome, as determined by electron microscopic analyses of polysomai RNA hybridized to single-or doublestranded DNA. We previously reported that eight different rightward-transcribed mRNAs contain, at their 5' ends, a common three-part leader sequence derived from coordinates 16.6,19.6 and 26.6, and that a ninth mRNA consists of sequences from 4.6-6.1 linked directly to those from 9.7-11.0 (Chow et al., 1977a; revised coordinates). We now demonstrate that four additional RNA species also have the same tripartite leader joined to messages with 5' ends located at coordinates at 29.1, 30.5, 33.9 and 42.6. The RNA transcript extending from 29.1-39.0 covers most or all of the viral associated (VA) RNA genes (29.0-30.1). The late RNAs can be grouped into several families of transcripts in which two or three mRNAs have the same 3' end and 3' proximal sequences, but differ substantially in length at the 5' end and, therefore, the coordinate at which the leader sequences are attached to the main body of the message. The number of RNA species transcribed from the Ad2 chromosome between coordinates 29 and 50 exceeds the number of identlfled Ad2 proteins. Several pairs of messages differing In length by a few percent may thus encode the same protein or have a precursor-product relationship. We have also found that a subpopulatlon of the polysomal mRNAs for the fiber protein, the most distal gene (66.3-91.2) in the rightward transcription unit, has a fourth leader component "y" (76.6-79.1) in addition to the common tripartite leader sequences. Less frequent fiber RNA species have fifth "x" (76.9-77.3) and/or sixth "z" (64.7-65.1) leader segments. Transcripts with x, y or z segments might be processing intermediates, and it is possible to arrange these species into an array suggestive of multiple pathways for the maturation of fiber mRNA. mRNA transcribed at late times from the L strand between coordinates 15.0 and 11.0 has a single component leader located 200 bases from the main body of the gene from coordinate 16.1 to 15.7. This leader originates from a site very close to the first leader component (16.5-16.6) of the R strand messages and suggests the presence of adjacent L and R strand promoters from which transcription diverges. Taken together, these results emphasize the diversity of splicing patterns and the varlety of recombined sequences generated during the synthesis of late Ad2 mRNAs.
The qnantitation and distribution of splicing intermediates in HeLa cells and adenovirus RNAs
Nucleic Acids Research, 1987
The steady state level of splicing intermediates in HeLa cells and in adenovirus RNA made late in the infectious cycle has been measured by a branch point analysis. About one in ten poly A(+) nuclear RNAs contained a branch point, but only 1/3 as many adenovirus RNAs were branched. Fewer branches were found in the poly A(-) RNAs of the nucleus and of late adenovirus transcripts suggesting that excised lariat introns do not accumulate in vivo. Branched RNAs were found in the poly A(+) RNAs from a nuclear maitrix fraction, but several experiments failed to show an enrichment in these splicing intermediates in this matrix fraction. Branches were found in all size classes of poly A(+) nuclear RNA and were not exclusively associated with either the 3' or 5' regions, but were randomly distributed within RNA molecules. These results as well as the base and sequence data on branch points (1,18) are consistent with the conclusion that branched poly A(+) RNAs are splicing intermediates.
An mRNA fraction coding for hexon polypep-tide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonu-clease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5! terminus were not hydrogen bonded to the single-stranded DNA. DNA se uences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17,20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of vira RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA. Most eukaryotic mRNAs bear modifications at both termini; their 3' termini have a tract of poly(A) that ranges in length from 30 to 200 bases (1-4), while their 5' termini are typically capped with a methylated guanine joined through a 5'-5' py-rophosphate linkage to a second nucleotide methylated at its 2' position (5, 6). Both types of modifications of eukaryotic mRNA are known to occur after transcription. All adenovirus mRNAs are thought to contain poly(A) tracts at their 3' termini (7) and be capped with a methylated guanine (8, 9). Specific restriction endonuclease cleavage fragments of adenovirus 2 (Ad2) DNA have permitted the mapping of regions of the genome expressed as mRNA and viral proteins during different stages of the lytic cycle (10-12). Little is known about the molecular mechanisms of viral mRNA synthesis. An important aspect of late mRNA synthesis is thought to be the processing and selection of viral mRNAs from the nucleus (18, 14). We have purified a late Ad2 hexon mRNA and found evidence providing some insight into the mechanism of synthesis of this mRNA. MATERIALS AND METHODS Isolation of Ad2 DNA and RNA. Polyribosomal RNA was prepared from Ad2-infected cells 32 hr after infection as described by Flint and Sharp (14, 15) and selected by chromatography on poly(U)-Sephadex (16). R-Loop Mapping. The R-loop hybridization mixture was essentially that of Thomas et al. (17) and contained 70% (vol/ vol) formamide [Matheson, Coleman, and Bell, 99%, further purified as described by Duesberg and Vogt (18)]; 0.20 M Tris-HCl, pH 7.91; 0.50 M NaCI; 0.01 M EDTA; Ad2 DNA at 10 ,g/ml; and purified hexon mRNA at 1-10 Ag/ml. This mixture was incubated at 52.50 for 2-3 hr and spread on a hy-The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3171 pophase of water with internal length standards of DNA from bacteriophage qX174, 5375 bases (19). Hybridization to Single-Stranded Ad2 DNA. Hybridiza-tions of either polyribosomal poly(A) or purified hexon mRNA with restriction endonuclease fragments of Ad2 DNA were carried out in reaction mixtures of-80% formamide; 0.40 M NaCl; 0.04 M 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.2; 0.01 M EDTA; DNA at 10 lg/ml; and hexon mRNA at 1.0-10 ,ug/ml (20). The sample was incubated at 57-60° for 2-3 hr. RESULTS Adenovirus late mRNAs begin to appear on polyribosomes about 13 hr after infection and continue to accumulate in the cell throughout the lytic cycle (21). Thus, to fractionate the most abundant late mRNAs, polyribosomes were prepared from cells 32 hr after infection with Ad2 and poly(A)-containing mRNA was selected by chromatography on poly(U)Sephadex columns. These mRNAs were then resolved into different molecular weight fractions by electrophoresis in 2.4-4.0% linear gradient polyacrylamide gels containing a uniform concentration of 7 M urea. After staining with ethidium bromide, distinct fluorescent bands were present in gels containing mRNA from virus-infected cells that were not found in gels containing identically prepared HeLa cell mRNA (Fig. 1A). These virus-specific RNAs were selectively labeled when [32P]phosphate was added to infected cells 24 hr after infection and the same mRNA fractions were prepared (Fig. 1B). RNA from the predominant ethidium bromide-staining band migrating 1.5 times faster than 28S rRNA in Fig. 1A and marked with a large arrow has been shown to code for the hexon polypeptide by in vitro translation (S. M. Berget, B. E. Roberts, and P. A. Sharp, data not shown). Furthermore, this RNA has been mapped by the R-loop technique (see below) to a region of the genome known to code for hexon (12) and is complementary to the r strand of the viral DNA (11). This mRNA species, therefore, will be referred to in the following sections as hexon mRNA. R-Loop Mapping of Hexon RNA. The R-loop technique developed by White and Hogness (22) and Thomas et al. (17) was used to position purified hexon mRNA on the viral genome. RNA eluted from a gel similar to that shown in Fig. IA was incubated, as described in Materials and Methods, with either total Ad2 DNA or restriction endonuclease fragments. Of the 43 total Ad2 DNA molecules scored as containing R-loops, 41 were observed to have a single region of hybrid, while two molecules contain a second R-loop, apparently in the region of the genome coding for the 1O(K polypeptide (12). Fig. 2 shows two examples of R-loops resulting from hy-bridization of hexon mRNA to fragments generated by the cleavage of Ad2 DNA with the EcoRI restriction endonuclease Abbreviation: Ad2, adenovirus 2. * We dedicate this work to the memory ofJerome Vinograd, a nan who loved science.
Influence of RNA Secondary Structure on the Pre-mRNA Splicing Process
Molecular and Cellular Biology, 2004
Pre-mRNA splicing in eukaryotes requires joining together the nucleotides of the various mRNA-coding regions (exons) after recognizing them from the normally vastly superior number of non-mRNA-coding sequences (introns). For three excellent reviews on general splicing and its regulation, refer to references 14, 62, and 70. In eukaryotes, the vast majority of splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate which has been estimated to contain several hundred different proteins in addition to five spliceosomal snRNAs . These factors are responsible for the accurate positioning of the spliceosome on the 5Ј and 3Ј splice site sequences. The reason why so many factors are needed reflects the observation that exon recognition can be affected by many pre-mRNA features such as exon length (5, 97), the presence of enhancer and silencer elements (8, 62), the strength of splicing signals (45), the promoter architecture (29, 55), and the rate of RNA processivity (86). In addition, the general cellular environment also exerts an effect, as recent observations suggest the existence of extensive coupling between splicing and many other gene expression steps (69) and even its modification by external stimuli (96).
The Journal of Biological Chemistry, 1987
Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.
Proceedings of the National Academy of Sciences, 2000
The AG dinucleotide at the 3 splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG 3 GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG 3 CG) or uracil (AG 3 UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG 3 HG). When adenine was replaced by a purine base (AG 3 PG) or by 7-deazaadenine (AG 3 c 7 AG), little effect on the second step was observed, suggesting that the 6-NH 2 and N 7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG 3 2-APG) had no effect on the second step. Taken together, our results suggest that the N 1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.
Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites
Molecular and Cellular Biology, 1984
The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a ...