scholarly journals Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

1989 ◽  
Vol 9 (1) ◽  
pp. 193-203 ◽  
Author(s):  
G Christofori ◽  
W Keller
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Cleavage Activity ◽  
Hela Cell Nuclear Extract ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Polyadenylation Factor ◽  
Polyadenylated Mrna ◽  
Cell Nuclear Extract

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro

1989 ◽  
Vol 9 (1) ◽  
pp. 193-203
Author(s):  
G Christofori ◽  
W Keller
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Cleavage Activity ◽  
Hela Cell Nuclear Extract ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Polyadenylation Factor ◽  
Polyadenylated Mrna ◽  
Cell Nuclear Extract

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

scholarly journals Mutations in poly(A) site downstream elements affect in vitro cleavage activity.

1988 ◽  
Vol 8 (4) ◽  
pp. 1839-1841 ◽  
Author(s):  
T L Green ◽  
R P Hart
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Early Gene ◽  
Sequence Element ◽  
Cleavage Activity ◽  
Sequence Recognition ◽  
Hela Cell Nuclear Extract ◽  
Cell Nuclear Extract

Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.

Mutations in poly(A) site downstream elements affect in vitro cleavage activity

1988 ◽  
Vol 8 (4) ◽  
pp. 1839-1841
Author(s):  
T L Green ◽  
R P Hart
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Early Gene ◽  
Sequence Element ◽  
Cleavage Activity ◽  
Sequence Recognition ◽  
Hela Cell Nuclear Extract ◽  
Cell Nuclear Extract

Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.

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

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.

scholarly journals Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

2001 ◽  
Vol 21 (14) ◽  
pp. 4604-4613 ◽  
Author(s):  
Gang Wang ◽  
Greg T. Cantin ◽  
Jennitte L. Stevens ◽  
Arnold J. Berk
Keyword(s):  
Hela Cell ◽  
Molecular Mass ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
High Molecular Mass ◽  
Multiprotein Complexes ◽  
Hela Cell Nuclear Extract ◽  
Cell Nuclear Extract

ABSTRACT A number of mammalian multiprotein complexes containing homologs ofSaccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (∼500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (∼2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ∼500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 × 105to 6 × 105 molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the ∼2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The ∼2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

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

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.

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

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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