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Archives of Biochemistry and Biophysics May 2005The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are...
The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.
Topics: Circular Dichroism; Hydrogen-Ion Concentration; Intercalating Agents; Kinetics; Osmolar Concentration; Platinum; Poly A; Poly A-U; Proflavine; RNA; Salts; Spectrum Analysis; Thermodynamics; Titrimetry
PubMed: 15850561
DOI: 10.1016/j.abb.2005.03.009 -
European Journal of Biochemistry Oct 1988To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of...
To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.
Topics: Animals; Carrier Proteins; Ducks; Globins; In Vitro Techniques; Poly A; Poly(A)-Binding Proteins; Protein Biosynthesis; RNA, Messenger; Rabbits; Reticulocytes; Ribonucleotides
PubMed: 2901956
DOI: 10.1111/j.1432-1033.1988.tb14309.x -
Methods in Enzymology 2008This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the...
This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the length of a poly(A) tail are presented: the poly(A) length assay, the ligation-mediated poly(A) test (LM-PAT), and the RNase H assay. The first two methods are PCR-based assays involving cDNA synthesis from an oligo(dT) primer. The third method involves removing the poly(A) tail from the mRNA of interest. A major obstacle to studying the enzymatic step of mammalian mRNA decay has been the inability to capture mRNA decay intermediates with structural impediments such as the poly(G) tract used in yeast. To overcome this, we combined a standard kinetic analysis of mRNA decay with a tetracycline repressor-controlled reporter with an Invader RNA assay. The Invader RNA assay is a simple, elegant assay for the quantification of mRNA. It is based on signal amplification, not target amplification, so it is less prone to artifacts than other methods for nucleic acid quantification. It is also very sensitive, able to detect attomolar levels of target mRNA. Finally, it requires only a short sequence for target recognition and quantitation. Therefore, it can be applied to determining the decay polarity of a mRNA by measuring the decay rates of different portions of that mRNA.
Topics: Animals; Humans; Mammals; Mutation; Plasmids; Poly A; Proteins; RNA Stability
PubMed: 19111191
DOI: 10.1016/S0076-6879(08)02624-4 -
Analytical Chemistry Jan 2020Rapid and efficient detection of tumor cells is one of the central challenges for modern analytical technology. In this paper, we report a polyadenine (poly(A))...
Rapid and efficient detection of tumor cells is one of the central challenges for modern analytical technology. In this paper, we report a polyadenine (poly(A)) tail-based strategy for ultrasensitive detection of tumor cells in aqueous solution with an electrochemical technique. Specifically, tumor cell-specific EpCAM aptamers without any modification can tightly bind on cell membranes and facilitate the subsequent introduction of multiple poly(A) tails via programmable terminal deoxynucleotidyl transferase (TdT)-mediated elongation. Meanwhile, since tumor cells bearing poly(A) tails can be easily adsorbed onto the surface of gold electrodes through a strong interaction between adenosines and gold, a highly amplified electrochemical signal can be obtained. Thus, by virtue of poly(A) tails, the proposed method allows the detection of as low as 3 cells mL. Compared with the previously reported methods for tumor cells detection, this poly(A)-based homogeneous electrochemical method needs just one enzyme and one aptamer without any modification and avoids the complex and time-consuming modification process of the working electrode, which holds great potential application in the future.
Topics: Cell Membrane; Electrochemical Techniques; Flow Cytometry; Hep G2 Cells; Humans; Optical Imaging; Poly A; Tumor Cells, Cultured
PubMed: 31850744
DOI: 10.1021/acs.analchem.9b04877 -
Genes & Development Sep 1989The addition of a poly(A) tail has been examined in a HeLa cell nuclear extract using SV40 late RNAs that end at or near the natural poly(A) site. We find that the...
The addition of a poly(A) tail has been examined in a HeLa cell nuclear extract using SV40 late RNAs that end at or near the natural poly(A) site. We find that the addition of a full-length, 200-nucleotide poly(A) tail occurs in two discrete phases. In the first phase, the addition of each adenosine is dependent on the highly conserved sequence AAUAAA. Mutations in that sequence result in an accumulation of products that contain 9 or fewer adenosine residues. In the second phase, poly(A) addition no longer requires AAUAAA but, instead, requires the oligo(A) primer synthesized during the first phase. Thus, RNAs carrying an AAUAAA mutation and a 3'-terminal oligo(A) segment are extended efficiently to full-length poly(A). The transition between phases occurs with the addition of the tenth adenosine residue. An activity exists that limits the length of poly(A) added in the extract to approximately 200 nucleotides. The two phases share at least one component and are likely to involve the same poly(A) polymerase.
Topics: HeLa Cells; Humans; Nucleotidyltransferases; Poly A; Polynucleotide Adenylyltransferase; RNA Processing, Post-Transcriptional; RNA, Messenger; Regulatory Sequences, Nucleic Acid
PubMed: 2575065
DOI: 10.1101/gad.3.9.1401 -
Nucleic Acids Research Dec 2003The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded...
The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.
Topics: Base Sequence; Binding Sites; Electrophoretic Mobility Shift Assay; Endoribonucleases; Escherichia coli Proteins; Exoribonucleases; Host Factor 1 Protein; Molecular Sequence Data; Nucleic Acid Conformation; Poly A; Polyribonucleotide Nucleotidyltransferase; RNA Processing, Post-Transcriptional; RNA, Messenger; Ribosomal Proteins; Substrate Specificity; Thermodynamics
PubMed: 14654705
DOI: 10.1093/nar/gkg915 -
Photochemical & Photobiological... Aug 2014Poly(A) has significant relevance to mRNA stability, protein synthesis and cancer biology. The ability of two phenothiazinium dyes azure A (AA) and azure B (AB) to bind...
Poly(A) has significant relevance to mRNA stability, protein synthesis and cancer biology. The ability of two phenothiazinium dyes azure A (AA) and azure B (AB) to bind single-stranded poly(A) was studied by spectroscopic and calorimetric techniques. Strong binding of the dyes and the higher affinity of AA over AB were ascertained from absorbance and fluorescence experiments. Significant perturbation of the circular dichroism spectrum of poly(A) in the presence of these molecules with formation of induced CD bands in the 300-700 nm region was observed. Strong emission polarization of the bound dyes and strong energy transfer from the adenine base pairs of poly(A) suggested intercalative binding to poly(A). Intercalative binding was confirmed from fluorescence quenching experiments and was predominantly entropy driven as evidenced from isothermal titration calorimetry data. The negative values of heat capacity indicated involvement of hydrophobic forces and enthalpy-entropy compensation suggested noncovalent interactions in the complexation for both the dyes. Poly(A) formed a self-assembled structure on the binding of both the dyes that was more favored under higher salt conditions. New insights in terms of spectroscopic and thermodynamic aspects into the self-structure formation of poly(A) by two new phenothiazinium dyes that may lead to structural and functional damage of mRNA are revealed from these studies.
Topics: Azure Stains; Calorimetry, Differential Scanning; Circular Dichroism; Energy Transfer; Fluorescence Polarization; Humans; Molecular Structure; Nucleic Acid Conformation; Photochemical Processes; Poly A; RNA Stability; Spectrometry, Fluorescence; Spectrophotometry; Static Electricity; Thermodynamics
PubMed: 24953877
DOI: 10.1039/c4pp00085d -
Methods in Molecular Biology (Clifton,... 2004In cells, the poly(A) tail stimulates translation from messenger RNAs bearing a cap structure or viral IRES elements. This 3' end-mediated stimulation of translation is...
In cells, the poly(A) tail stimulates translation from messenger RNAs bearing a cap structure or viral IRES elements. This 3' end-mediated stimulation of translation is not reflected in commonly used commercial cell-free translation systems prepared from rabbit reticulocytes or wheat germ. We describe a simple procedure to generate poly(A) tail-responsive translation extracts from HeLa cells. We suggest that this procedure should be adaptable to many animal cell lines.
Topics: Cell-Free System; Encephalomyocarditis virus; HeLa Cells; Humans; In Vitro Techniques; Peptide Chain Initiation, Translational; Poly A; Protein Biosynthesis; RNA Caps; RNA, Messenger; RNA, Viral; Ribosomes
PubMed: 14770005
DOI: 10.1385/1-59259-750-5:171 -
Methods in Molecular Biology (Clifton,... 2024In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes...
In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End-Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3' ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5' extension. The choice of the oligonucleotide 3' end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5' extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost.
Topics: RNA; RNA, Messenger; DNA, Complementary; DNA; DNA Primers; Poly A
PubMed: 37824067
DOI: 10.1007/978-1-0716-3481-3_7 -
Cold Spring Harbor Protocols Apr 2015Adenylation status has an important role in the regulation of mRNA metabolism: mRNAs are deadenylated before degradation, microRNAs (miRNAs) can cause deadenylation, and...
Adenylation status has an important role in the regulation of mRNA metabolism: mRNAs are deadenylated before degradation, microRNAs (miRNAs) can cause deadenylation, and the poly(A) length of certain mRNAs is regulated during development. This protocol describes methods that can be used to measure the poly(A) tail length of specific mRNAs. These include, in the order of increasing sensitivity, (1) northern blotting of intact and experimentally deadenylated mRNAs and (2) northern blotting of intact and experimentally deadenylated mRNA fragments that have been cleaved near the 3' end with RNase H. Highly sensitive polymerase chain reaction (PCR)-based approaches are also discussed.
Topics: Blotting, Northern; Poly A; Polyadenylation
PubMed: 25834263
DOI: 10.1101/pdb.prot081034