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1.
A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor. 总被引:5,自引:3,他引:2
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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. 相似文献
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Splicing of the adenovirus-2 E1A 13S mRNA requires a minimal intron length and specific intron signals. 总被引:10,自引:4,他引:6
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The adenovirus E1A region encodes three overlapping mRNAs, designated 9S, 12S and 13S. They differ from each other with regard to the length of the intron which is removed by RNA splicing. We have constructed E1A genes with deletions and insertions in the intervening sequence that is common to all three E1A mRNAs, in a search for signals which influence splicing of the 13S mRNA. Mutant plasmids were transfected into HeLa cells and the transiently expressed E1A mRNAs characterized by the S1 protection assay. The results show that five upstream and 20 downstream nucleotides are sufficient to allow for a correct utilization of the 5'-splice junction for the E1A 13S mRNA. Moreover, we show that a minimal intron length of 78 nucleotides is required for efficient 13S mRNA splicing. The ability of mutants with large intron deletions to maturate a 13S mRNA could partially be restored by expanding the intron length with phage lambda sequences. However, in no case was the normal splicing efficiency obtained with these mutants. In contrast, one mutant in which sequences from the authentic 13S mRNA intron were used to expand the intron expressed almost normal levels of 13S mRNA, thus suggesting that signals which specifically promote 13S mRNA splicing exist. 相似文献
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Splicing of adenovirus 2 early region 1A mRNAs is non-sequential 总被引:28,自引:0,他引:28
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Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus 总被引:11,自引:0,他引:11
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. 相似文献
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Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein. 总被引:21,自引:19,他引:2
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Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins. 相似文献
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Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli. 总被引:4,自引:0,他引:4
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The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG. 相似文献
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A Virtanen P Gilardi A N?slund J M LeMoullec U Pettersson M Perricaudet 《Journal of virology》1984,51(3):822-831
The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides. 相似文献
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Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector. 总被引:4,自引:2,他引:2
K Oda H Kato I Saito S Sugano K Maruyama M Masuda K Shiroki H Shimojo 《Journal of virology》1983,45(1):408-419
Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region. 相似文献
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Adenovirus mutants with splice-enhancing mutations in the E3 complex transcription unit are also defective in E3A RNA 3'-end formation 总被引:5,自引:3,他引:2
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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. 相似文献
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Zein genes, the genes coding for the zein storage proteins of maize, have a unique gene structure where at least two promoters lie upstream of the coding region. Between the P1 promoter (900 base pairs upstream of the coding region) and the translation initiation AUG codon are 18 short reading frames. A discrepancy between the signals obtained by S1-mapping and primer extension and the DNA sequence in the region of one of these signals suggests the presence of a 3' splice site lying 40 nucleotides upstream of the coding region. A splicing event removing all of the short reading frames from the mRNA transcribed from the P1 promoter would bring this mRNA into a translatable form. Further evidence for a functional 3' splice site has been obtained from sequencing of primer extension products and in vitro splicing of a hybrid intron in the HeLa cell in vitro splicing system. 相似文献
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Structure of three spliced mRNAs from region E3 of adenovirus type 2 总被引:13,自引:0,他引:13
A cDNA library representing early adenovirus type 2 (Ad2) mRNA was constructed. The cDNA copies were inserted into the PstI cleavage site of the pBR322 plasmid, and clones containing sequences from region E3 of the Ad2 genome were identified by colony hybridization. Selected clones were characterized by restriction enzyme cleavage, hybridization, and partial DNA sequence analysis. The precise structure of three spliced mRNAs was established by comparing the results with the DNA sequence of region E3 from Ad2 (Herissé et al., Nucl. Acids Res. 8 (1980) 2173--2191; Herissé and Galibert, Nucl. Acids Res. 9 (1981) 1229--1249). One of the characterized mRNA species encodes the E3/19K glycoprotein, whereas the other two most likely encode the E3/14K protein. The results demonstrate, moreover, that certain splice points which are used to generate the major E3 mRNAs are also used to splice the supplementary leader segments to the fibre mRNA at late times after infection. Two separate poly(A)-addition sites were identified in region E3 by analysis of the cDNA clones; one is preceded by the hexanucleotide sequence AAUAAA, whereas the other is preceded by an altered hexanucleotide, having the sequence AUUAAA. 相似文献
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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 总被引:3,自引:0,他引:3
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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. 相似文献
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Elimination of mRNA splicing by a point mutation outside the conserved GU at 5'' splice sites 总被引:9,自引:4,他引:5
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Nearly all mRNA introns begin with the dinucleotide GU. Mutations in either of these virtually invariant bases have been found to inactivate the corresponding 5' splice site. Until now single base changes in neighboring bases have not been found to completely inactivate a 5' splice site. Here we show that a single A----U transversion in the third position of the adenovirus 2 E1A 13S mRNA intron does prevent RNA splicing at the corresponding 5' splice site. 相似文献