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Molecular and Cellular Biology Apr 1988The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A)...
The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.
Topics: Adenine; Base Sequence; Cell Line; Cell-Free System; Humans; Kinetics; Nucleotide Mapping; Poly A; Proto-Oncogenes; RNA, Messenger; Uracil
PubMed: 3380094
DOI: 10.1128/mcb.8.4.1697-1708.1988 -
Bioinformatics (Oxford, England) Jan 2012Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying...
MOTIVATION
Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants.
CONTACT
vladimir.bajic@kaust.edu.sa
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Algorithms; Genome, Human; Humans; Internet; Neural Networks, Computer; Poly A; Sensitivity and Specificity; Software
PubMed: 22088842
DOI: 10.1093/bioinformatics/btr602 -
Philosophical Transactions of the Royal... Nov 2018Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of... (Review)
Review
Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin-antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.
Topics: Bacteria; Poly A; Polyadenylation; Prokaryotic Cells; RNA
PubMed: 30397102
DOI: 10.1098/rstb.2018.0166 -
RNA (New York, N.Y.) Jul 2005The poly(A)-limiting element (PLE) is a conserved sequence that restricts the length of the poly(A) tail to <20 nt. This study compared the translation of PLE-containing... (Comparative Study)
Comparative Study
The poly(A)-limiting element (PLE) is a conserved sequence that restricts the length of the poly(A) tail to <20 nt. This study compared the translation of PLE-containing short poly(A) mRNA expressed in cells with translation in vitro of mRNAs with varying length poly(A) tails. In transfected cells, PLE-containing mRNA had a <20-nt poly(A) and accumulated to a level 20% higher than a matching control without a PLE. It was translated as well as the matching control mRNA with long poly(A) and showed equivalent binding to polysomes. Translation in a HeLa cell cytoplasmic extract was used to examine the impact of the PLE in the context of varying length poly(A) tails. Here the overall translation of +PLE mRNA was less than control mRNA with the same length poly(A), and the PLE did not overcome the effect of a short poly(A) tail. Because poly(A)-binding protein (PABP) is a dominant effector of poly(A)-dependent translation we reasoned excess PABP in our extract might overwhelm PLE regulation of translation. This was confirmed by experiments where PABP was inactivated with poly(rA) or Paip2, and the effect of both treatments was reversed by addition of recombinant PABP. These data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA.
Topics: Animals; Base Sequence; COS Cells; Cell Culture Techniques; Chlorocebus aethiops; Conserved Sequence; HeLa Cells; Humans; Molecular Sequence Data; Poly A; Polyribosomes; Protein Biosynthesis; RNA, Messenger; RNA-Binding Proteins; Recombinant Proteins
PubMed: 15929942
DOI: 10.1261/rna.2470905 -
International Journal of Molecular... Dec 2019The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive...
The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive secondary species such as low-energy electrons (LEEs) with the most probable energy around 10 eV are generated, which are able to induce DNA strand breaks via dissociative electron attachment. Absolute DNA strand break cross sections of specific DNA sequences can be efficiently determined using DNA origami nanostructures as platforms exposing the target sequences towards LEEs. In this paper, we systematically study the effect of the oligonucleotide length on the strand break cross section at various irradiation energies. The present work focuses on poly-adenine sequences (d(A), d(A), d(A), d(A), and d(A)) irradiated with 5.0, 7.0, 8.4, and 10 eV electrons. Independent of the DNA length, the strand break cross section shows a maximum around 7.0 eV electron energy for all investigated oligonucleotides confirming that strand breakage occurs through the initial formation of negative ion resonances. When going from d(A) to d(A), the strand break cross section increases with oligonucleotide length, but only at 7.0 and 8.4 eV, i.e., close to the maximum of the negative ion resonance, the increase in the strand break cross section with the length is similar to the increase of an estimated geometrical cross section. For d(A), a markedly lower DNA strand break cross section is observed for all electron energies, which is tentatively ascribed to a conformational change of the dA sequence. The results indicate that, although there is a general length dependence of strand break cross sections, individual nucleotides do not contribute independently of the absolute strand break cross section of the whole DNA strand. The absolute quantification of sequence specific strand breaks will help develop a more accurate molecular level understanding of radiation induced DNA damage, which can then be used for optimized risk estimates in cancer radiation therapy.
Topics: DNA; DNA Damage; Electrons; Models, Chemical; Nanostructures; Oligonucleotides; Poly A
PubMed: 31877939
DOI: 10.3390/ijms21010111 -
Analytical Biochemistry Jul 2003Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high...
Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.
Topics: Animals; Archaeology; DNA; DNA Fragmentation; Dinucleotide Repeats; Genotype; History, Ancient; Humans; Microsatellite Repeats; Poly A; Polymerase Chain Reaction; Trinucleotide Repeats; Yugoslavia
PubMed: 12782040
DOI: 10.1016/s0003-2697(03)00160-x -
Inorganic Chemistry Jul 2017The first investigation of chiral ruthenium(II) complexes Δ- and Λ-[Ru(bpy)dppz] and triplex RNA poly(U)·poly(A)*poly(U) was carried out, which showed that Δ...
The first investigation of chiral ruthenium(II) complexes Δ- and Λ-[Ru(bpy)dppz] and triplex RNA poly(U)·poly(A)*poly(U) was carried out, which showed that Δ enantiomer displayed significant ability in stabilizing model triplex RNA.
Topics: Binding Sites; Molecular Conformation; Organometallic Compounds; Poly A; Poly U; RNA; Ruthenium
PubMed: 28636339
DOI: 10.1021/acs.inorgchem.7b00670 -
Methods in Molecular Biology (Clifton,... 2011Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according...
Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A) tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare samples for microarray analysis. RNA or crude lysate is mixed with biotinylated oligo(dT), which is then bound to paramagnetic streptavidin beads. Oligoadenylated mRNA is eluted first with a high salt buffer, followed by a low salt elution for polyadenylated mRNA. Elution of the RNA in two fractions can be used as a preparation of samples for microarray analysis while elution of the mRNA in several fractions can be used to analyse (changes in) poly(A) tail length. This method allows for accurate quantification of the amount of oligoadenylated/polyadenylated RNA in each fraction because it is not dependent on visualising the smears representing the variations in poly(A) tail length. The method is technically easy, fast, highly reproducible and can be performed on almost any sample containing RNA.
Topics: Chemical Fractionation; Microarray Analysis; Oligodeoxyribonucleotides; Poly A; Polyadenylation; RNA, Messenger; Streptavidin
PubMed: 21125487
DOI: 10.1007/978-1-59745-248-9_9 -
RNA (New York, N.Y.) Jul 2001The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of...
The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3' splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5' pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an approximately 62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.
Topics: Base Sequence; Exons; HeLa Cells; Humans; Introns; Molecular Sequence Data; Nuclear Proteins; Poly A; RNA Processing, Post-Transcriptional
PubMed: 11453064
DOI: 10.1017/s1355838201010329 -
Proceedings of the National Academy of... Oct 1985Fractionation of rat L6 myoblast histone H4 mRNA into its three component subspecies revealed that one of the major subspecies (H4-1) contained poly(A). The unique...
Fractionation of rat L6 myoblast histone H4 mRNA into its three component subspecies revealed that one of the major subspecies (H4-1) contained poly(A). The unique poly(A)+ H4 mRNA makes up about 8% of the total polysomal H4 mRNA population detected. Unlike the poly(A)- histone mRNAs, whose levels are reduced by greater than 95% when myoblasts differentiate into myotubes, the poly(A)+ subspecies is reduced by only 70%. The poly(A)+ H4 mRNA from myotubes incubated with actinomycin D decays with a half-life of 37-42 min, which is similar to that obtained for the poly(A)- H4 mRNAs in myoblasts. Both the poly(A)+ and poly(A)- subspecies decay at an increased rate after inhibition of DNA synthesis. In myoblasts the poly(A)+ H4 mRNA exists almost exclusively in the polysomal compartment (greater than 95%) with little (less than 5%) in the free ribonucleoprotein (mRNA-protein or mRNP) complex compartment of the cell. Poly(A)- histone H4 mRNA subspecies, on the other hand, are distributed with approximately 80% in the polysomal compartment and 20% in the free mRNP complex compartment. The unique poly(A)+ H4 mRNA is unusual, not only in that it contains poly(A) but also in its behavior compared to poly(A)- H4 mRNAs during terminal differentiation.
Topics: Animals; Base Sequence; Cell Line; Histones; Kinetics; Molecular Weight; Muscles; Poly A; RNA, Messenger; Rats
PubMed: 2864691
DOI: 10.1073/pnas.82.20.6760