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European Journal of Biochemistry Apr 1989ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for... (Comparative Study)
Comparative Study
ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and beta-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA-binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.
Topics: Animals; Blotting, Northern; Cell Nucleus; Energy Metabolism; Histones; Kinetics; Leukemia L5178; Leukemia, Experimental; Mice; Nuclear Envelope; Nucleic Acid Hybridization; Plasmids; Poly A; RNA, Messenger; Ribonucleotides; Transcription, Genetic
PubMed: 2565812
DOI: 10.1111/j.1432-1033.1989.tb14706.x -
Nucleic Acids Research Sep 2019Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more...
Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.
Topics: Benchmarking; Cell Line, Tumor; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Microfluidic Analytical Techniques; Poly A; RNA, Circular; RNA, Messenger; RNA, Ribosomal; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 31216024
DOI: 10.1093/nar/gkz535 -
Molecular and Cellular Biology Sep 1987The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal...
The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.
Topics: Binding Sites; Carrier Proteins; Cell Division; Cell Survival; DNA Mutational Analysis; Osmolar Concentration; Poly A; Poly(A)-Binding Proteins; Saccharomyces cerevisiae; Structure-Activity Relationship
PubMed: 3313012
DOI: 10.1128/mcb.7.9.3268-3276.1987 -
Genes & Development Jul 2016Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we...
Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ∼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.
Topics: Animals; Drosophila; Drosophila Proteins; Embryo, Nonmammalian; HeLa Cells; Humans; Oocytes; Poly A; Polyadenylation; Polynucleotide Adenylyltransferase; RNA, Messenger; Ribosomes; Sequence Analysis, RNA
PubMed: 27445395
DOI: 10.1101/gad.284802.116 -
Nature Chemistry Apr 2016The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA 'alphabet' by...
The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA 'alphabet' by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.
Topics: Circular Dichroism; DNA; Hydrogen Bonding; Peptide Nucleic Acids; Poly A; Triazines
PubMed: 27001733
DOI: 10.1038/nchem.2451 -
Chembiochem : a European Journal of... May 2012A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three...
A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N(6) pA can be visualized and purified by using click chemistry.
Topics: Adenosine; Click Chemistry; DNA-Directed RNA Polymerases; Gene Expression; HEK293 Cells; HeLa Cells; Humans; Poly A; RNA; RNA Polymerase II; RNA, Messenger
PubMed: 22513998
DOI: 10.1002/cbic.201200091 -
Nature Communications Jan 2021Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised....
Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3'-end processing in the maintenance of effective DDR.
Topics: Cell Cycle; Cell Line; DNA; DNA Damage; DNA Repair; DNA Replication; Gene Expression Regulation; HCT116 Cells; HeLa Cells; Histones; Humans; Jurkat Cells; Poly A; RNA, Messenger
PubMed: 33441544
DOI: 10.1038/s41467-020-20520-6 -
European Journal of Biochemistry May 1976Isoguanosine-5'-pyrosphosphate, in the presence of an oligonucleotide primer, was polymerized by Escherichia coli polynucleotide phosphorylase under conditions analogous...
Isoguanosine-5'-pyrosphosphate, in the presence of an oligonucleotide primer, was polymerized by Escherichia coli polynucleotide phosphorylase under conditions analogous to those required for polymerization of 5'-GMP. The resulting poly(isoguanylic acid), poly(isoG), was a multistranded helix with a stability considerably higher than that of poly(G), and fully resistant to various nucleolytic enzymes. The polymer exhibited a two-step temperature transition profile in moderately alkaline propylene glycol. Alkaline titration in aqueous medium, by ultraviolet and circular dichroism spectroscopy, showed two clearly defined transitions, the second of which was fully cooperative. The accompanying changes in sedimentation constants were consistent with a structure for poly(isoG) of a fourstranded helix, like neutral poly(G). In acid medium, spectral and potentiometric titrations demonstrated the existence of more than one transition in the pH range 6-12, with accompanying protonation of the isoguanosine residues. In neutral medium the polymer formed no complexes with other potentially complementary homopolymers. In acid medium, on the other hand, the protonated form of poly(isoG) did form a triple-stranded complex with poly(I), viz. 2poly(I) . poly(isoG)+. Possible structures are formulated for the neural and protonated forms of poly(isoG) which account for the two-step thermal transition in alkaline propylene glycol and on alkaline titration in aqueous medium. The nature of the protonated form, and its complex with poly(I) is also discussed.
Topics: Binding Sites; Circular Dichroism; Escherichia coli; Guanine; Kinetics; Nucleic Acid Conformation; Nucleic Acid Denaturation; Poly A; Poly G; Polyribonucleotide Nucleotidyltransferase; Polyribonucleotides; Potentiometry; Spectrophotometry, Ultraviolet; Temperature
PubMed: 776626
DOI: 10.1111/j.1432-1033.1976.tb10404.x -
The Journal of Biological Chemistry Aug 2009Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, approximately 250 nucleotides in mammalian cells. The same type of length control is...
Poly(A) tail length is controlled by the nuclear poly(A)-binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor.
Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, approximately 250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of approximately 250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of approximately 250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.
Topics: Animals; Cattle; Cleavage And Polyadenylation Specificity Factor; Electrophoretic Mobility Shift Assay; Poly A; Poly(A)-Binding Protein I; Polynucleotide Adenylyltransferase; Protein Binding; RNA 3' Polyadenylation Signals
PubMed: 19509282
DOI: 10.1074/jbc.M109.018226 -
The Journal of Biological Chemistry Jun 1997The 5'-cap and the poly(A) tail act synergistically to increase the translational efficiency of eukaryotic mRNAs, which suggests that these two mRNA elements communicate...
The 5'-cap and the poly(A) tail act synergistically to increase the translational efficiency of eukaryotic mRNAs, which suggests that these two mRNA elements communicate during translation. We report here that the cap-associated eukaryotic initiation factors (eIFs), i. e. the two isoforms of the cap-binding complex (eIF-4F and eIF-iso4F) and eIF-4B, bind to the poly(A)-binding protein (PABP) both in the presence and absence of poly(A) RNA. The interactions between PABP and eIF-4F, eIF-iso4F, and eIF-4B were measured in the absence of poly(A) RNA using far Western analysis and confirmed by direct fluorescence titration studies. The functional consequence of the interaction between these initiation factors and PABP was examined using RNA binding assays and RNA mobility shift analysis. eIF-4F, eIF-iso4F, and eIF-4B promoted PABP activity through a shift in its equilibrium affinity for poly(A). eIF-iso4G, the large subunit of eIF-iso4F, was the subunit responsible for the interaction between eIF-iso4F and PABP and was the subunit that promoted PABP RNA binding activity. Truncation analysis of eIF-iso4G indicated that a domain close to its N-terminal end appeared to be involved in binding PABP. These results suggest that the interaction between PABP and eIF-4B and eIF-iso4G may be involved in mediating the functional co-dependence observed between the cap and the poly(A) tail during translation.
Topics: Binding Sites; Eukaryotic Initiation Factor-4F; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Fluorescence; Peptide Initiation Factors; Poly A; Poly(A)-Binding Proteins; RNA, Messenger; RNA-Binding Proteins
PubMed: 9195926
DOI: 10.1074/jbc.272.26.16247