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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.
Mol Cell Biol 1992 Aug
PMID:Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA. 135 51

Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated. To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation. A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis. The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E. coli DNA template. A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E. coli, a gene that had originally been identified as controlling plasmid copy number [J. Lopilato, S. Bortner & J. Beckwith (1986) Mol. Gen. Genet. 205, 285-290] and was subsequently cloned and sequenced [J. Liu & J. S. Parkinson (1989) J. Bacteriol. 171, 1254-1261]. Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction. No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene. The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.
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PMID:Identification of the gene for an Escherichia coli poly(A) polymerase. 143 24

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.
Mol Cell Biol 1992 Jul
PMID:Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase. 162 Jan 31

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.
Mol Cell Biol 1990 Apr
PMID:Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA. 196 11

To elucidate the mechanism by which poly(A) polymerase functions in the 3'-end processing of pre-mRNAs, polyadenylation-specific RNP complexes were isolated by sedimentation in sucrose density gradients and the fractions were analyzed for the presence of the enzyme. At early stages of the reaction, the RNP complexes were resolved into distinct peaks which sedimented at approximately 18S and 25S. When reactions were carried out under conditions which support cleavage or polyadenylation, the pre-mRNA was specifically assembled into the larger 25S RNP complexes. Polyclonal antibodies raised against the enzyme purified from a rat hepatoma, which have been shown to inhibit cleavage and polyadenylation (Terns, M., and Jacob, S. T., Mol. Cell. Biol. 9:1435-1444, 1989) also prevented assembly of the 25S polyadenylation-specific RNP complexes. Furthermore, formation of these complexes required the presence of a chromatographic fraction containing poly(A) polymerase. UV cross-linking analysis indicated that the purified enzyme could be readily cross-linked to pre-mRNA but in an apparent sequence-independent manner. Reconstitution studies with the fractionated components showed that formation of the 25S RNP complex required the poly(A) polymerase fraction. Although the enzyme has not been directly localized to the specific complexes, the data presented in this report supports the role of poly(A) polymerase as an essential component of polyadenylation-specific complexes which functions both as a structural and enzymatic constituent.
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PMID:Potential role of poly(A) polymerase in the assembly of polyadenylation-specific RNP complexes. 201 73

Virtually all mRNAs in eucaryotes end in a poly(A) tail. This tail is added posttranscriptionally. In this report, we demonstrate that the enzyme that catalyzes this modification is identical with an activity first identified 30 years ago, the function of which was previously unknown. This enzyme, poly(A) polymerase, lacks any intrinsic specificity for its mRNA substrate but gains specificity by interacting with distinct molecules: a poly(A) polymerase from calf thymus, when combined with specificity factor(s) from cultured human cells, specifically and efficiently polyadenylates only appropriate mRNA substrates. Our results thus demonstrate that this polymerase is responsible for the addition of poly(A) to mRNAs and that its interaction with specificity factors is conserved.
Mol Cell Biol 1990 Feb
PMID:The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase. 215 26

In earlier studies the acute administration of tryptophan (TRP) to rats was reported to induce enhanced in vivo [14C]orotate-labeled hepatic nuclear RNA release in vitro. This change was considered to possibly be related to the induction of more and larger gamma-glutamyl transpeptidase-positive foci in the livers of rats treated with diethylnitrosamine and fed long-term elevated TRP in a choline-supplemented (CS) but not in a choline-deficient (CD) diet (comparisons with respective controls). In this study we investigated whether feeding a CD compared to a CS diet for 1 week would affect selected hepatic nuclear responses to TRP. Rats fed the CS but not the CD diet and tube-fed TRP 10 min before being killed revealed enhanced labeled hepatic nuclear RNA release in vitro. In all experiments, comparisons were made with the control groups (rats fed the CS or stock diet). When rats were fed elevated TRP (2%) in the diets (CS or CD) for 1 week, labeled hepatic nuclear RNA release was increased with the CS + TRP but not with the CD + TRP diet group. [3H]TRP binding to hepatic nuclei in vitro revealed no change in the CS + TRP group, decreased in the CD group, and markedly increased in the CD + TRP group in comparison with the control (CS) group. Hepatic nuclear nucleoside triphosphatase activity was increased only in the CS + TRP group while hepatic nuclear poly(A) polymerase activity was increased in the CS + TRP and in the CD +/- TRP groups. Serum cholesterol and triglycerides were decreased in the CD group and increased to control levels in the CD + TRP group.
Exp Mol Pathol 1989 Aug
PMID:Effect of feeding a choline-deficient diet on the hepatic nuclear response to tryptophan in the rat. 247 66

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.
Mol Cell Biol 1989 Jan
PMID:Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. 253 18

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.
Mol Cell Biol 1989 Apr
PMID:Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor. 256 10

To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.
Mol Cell Biol 1985 Feb
PMID:RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro. 257 21


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