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Genes & Development Jun 1989Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To...
Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.
Topics: Amino Acid Sequence; Animals; Base Sequence; Female; Gene Expression Regulation; Molecular Sequence Data; Oocytes; Oogenesis; Poly A; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Messenger; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Xenopus laevis
PubMed: 2568313
DOI: 10.1101/gad.3.6.803 -
Proceedings of the National Academy of... Jun 2000In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable. The selective susceptibility of...
In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable. The selective susceptibility of unstable Escherichia coli RNAs to 3' polyadenylation by the pcnB gene product, poly(A) polymerase I (PAP I), in vivo is a key factor in their rapid degradation by 3' to 5' exonucleases. Using highly purified His-tagged recombinant PAP I, we show that differential adenylation of RNA substrates by PAP I occurs in vitro and that this capability resides in PAP I itself rather than in any ancillary protein(s). Surprisingly, the efficiency of 3' polyadenylation is affected by substrate structure at both termini; single-strand segments at either the 5' or 3' end of RNA molecules and monophosphorylation at an unpaired 5' terminus dramatically increase the rate and length of 3' poly(A) tail additions by PAP I. Our results provide a mechanistic basis for the susceptibility of certain RNAs to 3' polyadenylation. They also suggest a model of "programmed" RNA decay in which endonucleolytically generated RNA fragments containing single-stranded monophosphorylated 5' termini are targeted for poly(A) addition and further degradation.
Topics: Escherichia coli; Phosphorylation; Poly A; Polynucleotide Adenylyltransferase; RNA
PubMed: 10823925
DOI: 10.1073/pnas.120173797 -
Molecular and Cellular Biology Jan 1989We 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...
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.
Topics: Cell Extracts; Cells, Cultured; Chemical Fractionation; Chromatography; HeLa Cells; Humans; Nucleotidyltransferases; Plasmids; Poly A; Polynucleotide Adenylyltransferase; RNA Precursors; RNA Splicing; RNA, Messenger; RNA, Nuclear
PubMed: 2538718
DOI: 10.1128/mcb.9.1.193-203.1989 -
The Journal of Biological Chemistry Oct 2003Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. An unsolved problem is how the poly(A) signal triggers the...
Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. An unsolved problem is how the poly(A) signal triggers the polymerase to terminate. A popular model is that this occurs during extrusion of the poly(A) signal, at which time it interacts with factors on the transcription complex. To test this idea we used cis-antisense inhibition in vivo to probe the temporal relationship between poly(A) signal extrusion and the commitment of the polymerase to terminate. Our rationale was to inactivate the poly(A) signal at increasing times post-extrusion to determine the point beyond which it is no longer required for termination. We found that communication with the polymerase is not temporally restricted to the time of poly(A) signal extrusion, but is ongoing and perhaps random. Some polymerases terminate almost immediately. Others have yet to receive their termination instructions from the poly(A) signal even 500 bp downstream, as indicated by the ability of an antisense at this distance to block termination. Thus, the poly(A) signal can functionally interact with the polymerase at considerable distances down the template. This is consistent with the emerging picture of a processing apparatus that assembles and matures while riding with the polymerase.
Topics: Animals; COS Cells; Macromolecular Substances; Models, Genetic; Plasmids; Poly A; RNA Polymerase II; RNA, Antisense; Signal Transduction; Time Factors; Transcription, Genetic; Transfection
PubMed: 12933817
DOI: 10.1074/jbc.M306304200 -
Nucleic Acids Research Jun 1989Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini...
Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.
Topics: Animals; Binding, Competitive; Cell Nucleus; Cytidine Deaminase; DNA Nucleotidylexotransferase; Exonucleases; Kinetics; Poly A; RNA; RNA Processing, Post-Transcriptional; RNA, Messenger; Trypanosoma cruzi
PubMed: 2473439
DOI: 10.1093/nar/17.12.4647 -
Molecular and Cellular Biology Jun 1988We developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of...
We developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region. Using this method, we studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin mRNA in hydrated rats was, quite remarkably, approximately 250 nucleotides in length, in contrast to that of somatostatin mRNA, which was approximately 30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from approximately 250 to approximately 400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation.
Topics: Animals; Drinking; Electrophoresis, Polyacrylamide Gel; Endoribonucleases; Hypothalamus; Nucleic Acid Hybridization; Poly A; RNA, Messenger; Rats; Ribonuclease H; Vasopressins; Water Deprivation
PubMed: 2841576
DOI: 10.1128/mcb.8.6.2267-2274.1988 -
European Journal of Biochemistry Feb 1976Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of... (Comparative Study)
Comparative Study
Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.
Topics: Animals; Base Sequence; Cell Nucleus; Kinetics; Liver; Male; Molecular Weight; Nucleic Acid Hybridization; Nucleic Acid Renaturation; Poly A; RNA; Xenopus
PubMed: 1253796
DOI: 10.1111/j.1432-1033.1976.tb10174.x -
The Journal of Biological Chemistry May 1981To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated...
To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.
Topics: Animals; Base Sequence; Kidney; Kinetics; Male; Mice; Molecular Weight; Nucleic Acid Hybridization; Poly A; Protein Biosynthesis; RNA, Messenger
PubMed: 6112224
DOI: No ID Found -
The Journal of Biological Chemistry May 2019Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3....
Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.
Topics: Humans; Peptide Chain Termination, Translational; Peptide Initiation Factors; Peptide Termination Factors; Poly A; RNA, Messenger; RNA-Binding Proteins; Repressor Proteins
PubMed: 30992367
DOI: 10.1074/jbc.RA118.006856 -
Proceedings of the National Academy of... Jul 1979The reovirus oligoadenylates exist in two states within the virion: free and bound to viral proteins. The latter class of oligonucleotides, after digestion with...
The reovirus oligoadenylates exist in two states within the virion: free and bound to viral proteins. The latter class of oligonucleotides, after digestion with Penicillium (P1) nuclease, yields adenylic acid and an adenosine-containing compound that is positively charged at pH 1.7, 3.5, or 6.5. In a mixture of [35S]methionine- and [3H]adenosine-labeled reovirus disrupted by sodium dodecyl sulfate/urea, approximately 4% of the radioactivity in [35S]methionine-labeled proteins coelutes with [3H]adenosine-labeled material at a net charge of -1.5 when analyzed by ion-exchange chromatography on DEAE-cellulose. This material migrates in sodium dodecyl sulfate/polyacrylamide gels with mu polypeptides and with a small protein, viii. Radioactivity is not released when the complex is boiled in buffer containing sodium dodecyl sulfate and urea or boiled in 80% dimethyl sulfoxide or when viral RNA is extracted with phenol. Digestion with Pronase converts the [3H]adenosine-labeled compound to oligomers of net charge -8 to -12 which contain nuclease P1- and alkaline phosphatase-sensitive adenylic acid residues as well as adenosine in a P1- and phosphatase-resistant linkage. These data indicate that reovirus contains structural proteins that are covalently bound to an oligoadenylate moiety.
Topics: Mammalian orthoreovirus 3; Oligoribonucleotides; Poly A; RNA, Viral; Reoviridae; Viral Proteins
PubMed: 290987
DOI: 10.1073/pnas.76.7.3087