In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.

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The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2317-2323.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2317-2323

Author(s):

D Zarkower ◽

P Stephenson ◽

M Sheets ◽

M Wickens

Keyword(s):

Hela Cell ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Polyadenylation Site ◽

Single Base ◽

Cleavage And Polyadenylation ◽

Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

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The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2317 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2317-2323 ◽

Cited By ~ 62

Author(s):

D Zarkower ◽

P Stephenson ◽

M Sheets ◽

M Wickens

Keyword(s):

Hela Cell ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Polyadenylation Site ◽

Single Base ◽

Cleavage And Polyadenylation ◽

Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

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A novel protein factor is required for use of distal alternative 5' splice sites in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5945-5953.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5945-5953

Author(s):

J E Harper ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Deae Cellulose ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

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Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.495 ◽

1987 ◽

Vol 7 (1) ◽

pp. 495-503 ◽

Cited By ~ 18

Author(s):

L C Ryner ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Nuclear Extract ◽

Structural Features ◽

Simian Virus ◽

Micrococcal Nuclease ◽

Small Nuclear Ribonucleoprotein ◽

Cleavage And Polyadenylation ◽

Nuclear Ribonucleoprotein ◽

Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

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A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.259-267.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 259-267

Author(s):

C H Agris ◽

M E Nemeroff ◽

R M Krug

Keyword(s):

Specific Protein ◽

Viral Mrna ◽

Sequence Element ◽

Time Points ◽

Total Absence ◽

Nuclear Extracts ◽

Infected Cells ◽

Small Nuclear Ribonucleoproteins ◽

Rna Precursor

The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the U1, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' and branch point of NS1 and mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 is controlled in infected cells.

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Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.495-503.1987 ◽

1987 ◽

Vol 7 (1) ◽

pp. 495-503

Author(s):

L C Ryner ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Nuclear Extract ◽

Structural Features ◽

Simian Virus ◽

Micrococcal Nuclease ◽

Small Nuclear Ribonucleoprotein ◽

Cleavage And Polyadenylation ◽

Nuclear Ribonucleoprotein ◽

Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

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Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4484 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4484-4491 ◽

Cited By ~ 17

Author(s):

A Mayeda ◽

Y Ohshima

Keyword(s):

Consensus Sequence ◽

Donor Site ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

T Antigen ◽

Beta Globin ◽

Vitro Assay ◽

Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

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Specific protein binding to the simian virus 40 enhancer in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2098 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2098-2105 ◽

Cited By ~ 49

Author(s):

A G Wildeman ◽

M Zenke ◽

C Schatz ◽

M Wintzerith ◽

T Grundström ◽

...

Keyword(s):

Point Mutations ◽

Simian Virus 40 ◽

Simian Virus ◽

Specific Protein ◽

Dnase I ◽

Wild Type ◽

Dnase I Footprinting ◽

Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

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Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.2051-2060.1985 ◽

1985 ◽

Vol 5 (8) ◽

pp. 2051-2060

Author(s):

B W Stillman ◽

Y Gluzman

Keyword(s):

Dna Synthesis ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

T Antigen ◽

Cell Extracts ◽

293 Cells ◽

Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

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