scholarly journals Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.

1988 ◽  
Vol 8 (5) ◽  
pp. 2052-2062 ◽  
Author(s):  
J E Stefano ◽  
D E Adams
Keyword(s):  
Adenovirus Type ◽  
Nuclear Extract ◽  
Rnase H ◽  
Specific Factor ◽  
Cell Nuclei ◽  
Sequence Element ◽  
Initial Event ◽  
Cleavage And Polyadenylation ◽  
A Site

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro

1988 ◽  
Vol 8 (5) ◽  
pp. 2052-2062
Author(s):  
J E Stefano ◽  
D E Adams
Keyword(s):  
Adenovirus Type ◽  
Nuclear Extract ◽  
Rnase H ◽  
Specific Factor ◽  
Cell Nuclei ◽  
Sequence Element ◽  
Initial Event ◽  
Cleavage And Polyadenylation ◽  
A Site

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

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 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.

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.

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.

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.

scholarly journals Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box.

1994 ◽  
Vol 14 (3) ◽  
pp. 2191-2200 ◽  
Author(s):  
J M Li ◽  
R A Parsons ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Rna Polymerase Iii ◽  
Nuclear Extract ◽  
Major Protein ◽  
In Vitro Mutagenesis ◽  
Flanking Sequence ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.

scholarly journals Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

1992 ◽  
Vol 12 (4) ◽  
pp. 1460-1468 ◽  
Author(s):  
C Wersig ◽  
A Bindereif
Keyword(s):  
Mutational Analysis ◽  
Nuclear Extract ◽  
Small Nuclear Rna ◽  
Interaction Domain ◽  
Sequence Element ◽  
Spliceosome Assembly ◽  
Small Nuclear Ribonucleoprotein ◽  
U4 Rna ◽  
Nuclear Ribonucleoprotein

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

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