Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor

1989 ◽  
Vol 9 (4) ◽  
pp. 1435-1444
Author(s):  
M P Terns ◽  
S T Jacob
Keyword(s):  
Cleavage Site ◽  
Cleavage Reaction ◽  
Endonucleolytic Cleavage ◽  
Nuclear Extracts ◽  
Adenylic Acid ◽  
Specific Complex ◽  
Functional Complex ◽  
Cleavage And Polyadenylation ◽  
Mrna Precursor

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.

scholarly journals Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

1989 ◽  
Vol 9 (4) ◽  
pp. 1435-1444 ◽  
Author(s):  
M P Terns ◽  
S T Jacob
Keyword(s):  
Cleavage Site ◽  
Cleavage Reaction ◽  
Endonucleolytic Cleavage ◽  
Nuclear Extracts ◽  
Adenylic Acid ◽  
Specific Complex ◽  
Functional Complex ◽  
Cleavage And Polyadenylation ◽  
Mrna Precursor

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly

1989 ◽  
Vol 9 (4) ◽  
pp. 1759-1771
Author(s):  
L C Ryner ◽  
Y Takagaki ◽  
J L Manley
Keyword(s):  
Cleavage Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Reaction ◽  
Mrna Cleavage ◽  
Nuclear Extracts ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Signals

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

scholarly journals Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

1989 ◽  
Vol 9 (4) ◽  
pp. 1759-1771 ◽  
Author(s):  
L C Ryner ◽  
Y Takagaki ◽  
J L Manley
Keyword(s):  
Cleavage Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Reaction ◽  
Mrna Cleavage ◽  
Nuclear Extracts ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Signals

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

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.

scholarly journals RBBP6 activates the pre-mRNA 3′-end processing machinery in humans

2021 ◽  
Author(s):  
Vytaute Boreikaite ◽  
Thomas Elliot ◽  
Jason Chin ◽  
Lori A Passmore
Keyword(s):  
Rna Processing ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Endonucleolytic Cleavage ◽  
Mature Transcript ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Additional Protein ◽  
Stimulatory Factor

3′-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3′-endonuclease activity of human CPSF with purified proteins. This requires the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

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 Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1092
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Stephen Oroszlan ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Cleavage Site ◽  
Potential Role ◽  
Binding Mode ◽  
Interaction Energies ◽  
Vitro Assay ◽  
Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

scholarly journals Long-range RNA interaction of two sequence elements required for endonucleolytic cleavage of human insulin-like growth factor II mRNAs.

1995 ◽  
Vol 15 (1) ◽  
pp. 235-245 ◽  
Author(s):  
W Scheper ◽  
D Meinsma ◽  
P E Holthuizen ◽  
J S Sussenbach
Keyword(s):  
Growth Factor ◽  
Long Range ◽  
Cleavage Site ◽  
Untranslated Region ◽  
Cleavage Product ◽  
Coding Region ◽  
Endonucleolytic Cleavage ◽  
Factor Ii ◽  
Sequence Elements ◽  
Rna Interaction

Human insulin-like growth factor II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region, leading to an unstable 5' cleavage product containing the IGF-II coding region and a very stable 3' cleavage product of 1.8 kb. This endonucleolytic cleavage is most probably the first and rate-limiting step in degradation of IGF-II mRNAs. Two sequence elements within the 3' untranslated region are required for cleavage: element I, located approximately 2 kb upstream of the cleavage site, and element II, encompassing the cleavage site itself. We have identified a stable double-stranded RNA stem structure (delta G = -100 kcal/mol [418.4 kJ/mol]) that can be formed between element I and a region downstream of the cleavage site in element II. This structure is conserved among human, rat, and mouse mRNAs. Detailed analysis of the requirements for cleavage shows that the relative position of the elements is not essential for cleavage. Furthermore, the distance between the coding region and the cleavage site does not affect the cleavage reaction. Mutational analysis of the long-range RNA-RNA interaction shows that not only the double-stranded character but also the sequence of the stable RNA stem is important for cleavage.

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