scholarly journals Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

1985 ◽  
Vol 5 (11) ◽  
pp. 2975-2983 ◽  
Author(s):  
R P Hart ◽  
M A McDevitt ◽  
H Ali ◽  
J R Nevins
Keyword(s):  
Essential Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Sites ◽  
Multiple Sequence ◽  
Polyadenylic Acid ◽  
Sequence Elements ◽  
Cleavage And Polyadenylation ◽  
Definition Of ◽  
A Site

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites

1985 ◽  
Vol 5 (11) ◽  
pp. 2975-2983
Author(s):  
R P Hart ◽  
M A McDevitt ◽  
H Ali ◽  
J R Nevins
Keyword(s):  
Essential Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Sites ◽  
Multiple Sequence ◽  
Polyadenylic Acid ◽  
Sequence Elements ◽  
Cleavage And Polyadenylation ◽  
Definition Of ◽  
A Site

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

Regulation of SV40 early gene expression

1988 ◽  
Vol 66 (6) ◽  
pp. 567-577 ◽  
Author(s):  
Alan G. Wildeman
Keyword(s):  
Transcriptional Control ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Site Directed Mutagenesis ◽  
Early Gene ◽  
Upstream Sequence ◽  
Sv40 Promoter ◽  
Multiple Sequence ◽  
Sequence Element ◽  
Sequence Elements

The early promoter of the simian virus 40 (SV40) has been used as a model eukaryotic promoter for the study of DN A sequence elements and cellular factors that are involved in transcriptional control and initiation. Site-directed mutagenesis and cell-free transcription systems have enabled the dissection of the functional domains within the 21 bp upstream sequence element and the 72 bp enhancer, and a number of protein factors that bind to various "motifs" within these domains have been identified. This article summarizes recent observations that have led to the conclusion that the SV40 promoter, and particularly, the enhancer region, is composed of multiple sequence elements. Some of these elements are present in cellular genes, and may exhibit tissue-specificity in their action. Furthermore, the proteins that are being identified (e.g., Sp1) may have binding sites within these elements that are sufficiently specific to ensure that only certain sets of genes will be selectively expressed.

scholarly journals Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs.

1987 ◽  
Vol 7 (4) ◽  
pp. 1518-1529 ◽  
Author(s):  
M D Sheets ◽  
P Stephenson ◽  
M P Wickens
Keyword(s):  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Hydroxyl Group ◽  
Endonucleolytic Cleavage ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Mrna Precursor ◽  
Crude Nuclear Extract

Formation of mRNA 3' termini involves cleavage of an mRNA precursor and polyadenylation of the newly formed end. Cleavage of simian virus 40 late pre-mRNA in a crude nuclear extract generated two RNAs, 5' and 3' half-molecules. These RNAs were unmodified and linear. The 5' half-molecule contained sequences upstream but not downstream of the poly(A) site and ended in a 3'-terminal hydroxyl. The 3' half-molecules comprised a family of RNAs, each of which contains only sequences downstream of the poly(A) site, and ends in a 5'-terminal phosphate. These RNAs differed only in the locations of their 5' terminus. The 3' terminus of the 5' half-molecule was the adenosine 10 nucleotides downstream of AAUAAA, at the +1 position. The 5' terminus of the longest 3' half-molecule was at +2. Thus, these two RNAs contain every nucleoside and phosphate of the precursor. The existence of these half-molecules demonstrates that endonucleolytic cleavage occurs near the poly(A) site. 5' half-molecules generated in the presence of EDTA (which blocks polyadenylation, but not cleavage) ended at the adenosine at position +1 of the precursor. When incubated in the extract under suitable conditions, they became polyadenylated. 5' half-molecules formed in 3'-dATP-containing reactions contained a single 3'-deoxyadenosine (cordycepin) residue added onto the +1 adenosine and were poor polyadenylation substrates. We infer that the +1 adenosine of the precursor becomes the first A of the poly(A) tract and provides a 3' hydroxyl group to which poly(A) is added posttranscriptionally.

Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs

1987 ◽  
Vol 7 (4) ◽  
pp. 1518-1529
Author(s):  
M D Sheets ◽  
P Stephenson ◽  
M P Wickens
Keyword(s):  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Hydroxyl Group ◽  
Endonucleolytic Cleavage ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Mrna Precursor ◽  
Crude Nuclear Extract

Formation of mRNA 3' termini involves cleavage of an mRNA precursor and polyadenylation of the newly formed end. Cleavage of simian virus 40 late pre-mRNA in a crude nuclear extract generated two RNAs, 5' and 3' half-molecules. These RNAs were unmodified and linear. The 5' half-molecule contained sequences upstream but not downstream of the poly(A) site and ended in a 3'-terminal hydroxyl. The 3' half-molecules comprised a family of RNAs, each of which contains only sequences downstream of the poly(A) site, and ends in a 5'-terminal phosphate. These RNAs differed only in the locations of their 5' terminus. The 3' terminus of the 5' half-molecule was the adenosine 10 nucleotides downstream of AAUAAA, at the +1 position. The 5' terminus of the longest 3' half-molecule was at +2. Thus, these two RNAs contain every nucleoside and phosphate of the precursor. The existence of these half-molecules demonstrates that endonucleolytic cleavage occurs near the poly(A) site. 5' half-molecules generated in the presence of EDTA (which blocks polyadenylation, but not cleavage) ended at the adenosine at position +1 of the precursor. When incubated in the extract under suitable conditions, they became polyadenylated. 5' half-molecules formed in 3'-dATP-containing reactions contained a single 3'-deoxyadenosine (cordycepin) residue added onto the +1 adenosine and were poor polyadenylation substrates. We infer that the +1 adenosine of the precursor becomes the first A of the poly(A) tract and provides a 3' hydroxyl group to which poly(A) is added posttranscriptionally.

Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site

1984 ◽  
Vol 4 (8) ◽  
pp. 1460-1468
Author(s):  
M Sadofsky ◽  
J C Alwine
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Site ◽  
Deletion Mutants ◽  
Processing Signal ◽  
Cleavage And Polyadenylation ◽  
Infected Cells ◽  
Polyadenylation Sites ◽  
Definition Of ◽  
Late Transcript

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.

scholarly journals Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site.

1984 ◽  
Vol 4 (8) ◽  
pp. 1460-1468 ◽  
Author(s):  
M Sadofsky ◽  
J C Alwine
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Site ◽  
Deletion Mutants ◽  
Processing Signal ◽  
Cleavage And Polyadenylation ◽  
Infected Cells ◽  
Polyadenylation Sites ◽  
Definition Of ◽  
Late Transcript

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.

scholarly journals Assembly of the Cleavage and Polyadenylation Apparatus Requires About 10 Seconds In Vivo and Is Faster for Strong than for Weak Poly(A) Sites

1999 ◽  
Vol 19 (8) ◽  
pp. 5588-5600 ◽  
Author(s):  
Lily C. Chao ◽  
Amer Jamil ◽  
Steven J. Kim ◽  
Lisa Huang ◽  
Harold G. Martinson
Keyword(s):  
Size Effect ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Antisense Inhibition ◽  
Assembly Process ◽  
Antisense Sequence ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Duplex Formation

ABSTRACT We have devised a cis-antisense rescue assay of cleavage and polyadenylation to determine how long it takes the simian virus 40 (SV40) early poly(A) signal to commit itself to processing in vivo. An inverted copy of the poly(A) signal placed immediately downstream of the authentic one inhibited processing by means of sense-antisense duplex formation in the RNA. The antisense inhibition was gradually relieved when the inverted signal was moved increasing distances downstream, presumably because cleavage and polyadenylation occur before the polymerase reaches the antisense sequence. Antisense inhibition was unaffected when the inverted signal was moved upstream. Based on the known rate of transcription, we estimate that the cleavage-polyadenylation process takes between 10 and 20 s for the SV40 early poly(A) site to complete in vivo. Relief from inhibition occurred earlier for shorter antisense sequences than for longer ones. This indicates that a brief period of assembly is sufficient for the poly(A) signal to shield itself from a short (50- to 70-nucleotide) antisense sequence but that more assembly time is required for the signal to become immune to the longer ones (∼200 nucleotides). The simplest explanation for this target size effect is that the assembly process progressively sequesters more and more of the RNA surrounding the poly(A) signal up to a maximum of about 200 nucleotides, which we infer to be the domain of the mature apparatus. We compared strong and weak poly(A) sites. The SV40 late poly(A) site, one of the strongest, assembles several times faster than the weaker SV40 early or synthetic poly(A) site.

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.

Multiple Sequence Elements Involved in Polyadenylation at The Murine Igm Secretory Poly(A)Site.

1997 ◽  
Vol 16 (5-6) ◽  
pp. 737-742
Author(s):  
Cathy Phillips ◽  
Anders Virtanen
Keyword(s):  
Multiple Sequence ◽  
Sequence Elements ◽  
A Site
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