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Nucleic Acids Research Apr 2019Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length....
Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length. These mechanisms play crucial roles in the dynamics of cell polarization and embryonic development. Furthermore, mRNAs are emerging therapeutics and chemical alterations to increase their translational efficiency are highly sought after. We show that yeast poly(A) polymerase can be used to install multiple azido-modified adenosine nucleotides to luciferase and eGFP-mRNAs. These mRNAs can be efficiently reacted in a bioorthogonal click reaction with fluorescent reporters without degradation and without sequence alterations in their coding or untranslated regions. Importantly, the modifications in the poly(A) tail impact positively on the translational efficiency of reporter-mRNAs in vitro and in cells. Therefore, covalent fluorescent labeling at the poly(A) tail presents a new way to increase the amount of reporter protein from exogenous mRNA and to label genetically unaltered and translationally active mRNAs.
Topics: Adenosine Triphosphate; Cell Survival; Fluorescence; HeLa Cells; Humans; Poly A; Protein Biosynthesis; RNA, Messenger; Staining and Labeling
PubMed: 30726958
DOI: 10.1093/nar/gkz084 -
Nucleic Acids Research Jan 2015Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3'-processing sites depending on the cell and tissue type and/or physiological...
Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3'-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3'-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3'-untranslated regions (3'-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish.
Topics: Animals; Databases, Nucleic Acid; Gene Expression; Humans; Internet; Mice; Poly A; Polyadenylation; RNA Cleavage; Zebrafish
PubMed: 25378337
DOI: 10.1093/nar/gku1076 -
Cell Jun 1992
Review
Topics: Animals; Biological Evolution; Meiosis; Oocytes; Poly A; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 1606613
DOI: 10.1016/0092-8674(92)90606-d -
RNA (New York, N.Y.) May 2022During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The...
During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding cleavage sites for over 2600 genes. We find that uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in .
Topics: 3' Untranslated Regions; Animals; Giardia lamblia; Mammals; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35110372
DOI: 10.1261/rna.078793.121 -
The Journal of Biological Chemistry Nov 2007Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that...
Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.
Topics: Adenine; Amino Acid Motifs; Animals; Binding Sites; Conserved Sequence; Exoribonucleases; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Poly A; Protein Binding; Protein Folding; Protein Structure, Tertiary; RNA; Sequence Alignment
PubMed: 17785461
DOI: 10.1074/jbc.M702375200 -
Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.Nature Communications Jan 2018Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the...
Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.
Topics: Animals; Cell Line, Tumor; Cells, Cultured; Ganglia, Spinal; Humans; Mice; Neurons; Nociceptive Pain; Pain; Pain Measurement; Poly A; Poly(A)-Binding Proteins; Protein Binding; RNA
PubMed: 29295980
DOI: 10.1038/s41467-017-02449-5 -
Annals of Medicine 2008The circadian clock is a conserved internal timekeeping mechanism that controls many aspects of physiology and behavior via the rhythmic expression of many genes. One of... (Review)
Review
The circadian clock is a conserved internal timekeeping mechanism that controls many aspects of physiology and behavior via the rhythmic expression of many genes. One of these rhythmic genes, Nocturnin, encodes a deadenylase--a ribonuclease that specifically removes the poly(A) tails from mRNAs. This enzyme is expressed at high levels during the night in a number of tissues in mammals and has recently been implicated in circadian control of metabolism. Targeted ablation of this gene in mice results in resistance to hepatic steatosis and diet-induced obesity. Nocturnin appears to exert rhythmic posttranscriptional control of genes necessary for metabolic functions including nutrient absorption, glucose/insulin sensitivity, and lipid storage. In the Western world and many developing countries, overnutrition--the 'obesity epidemic' suggests that the ability to sequester fat stores in times of plenty is no longer advantageous to our survival. Understanding the role that the circadian clock plays in controlling these metabolic processes is important in treatment and eventual eradication of this public health crisis.
Topics: Animals; Circadian Rhythm; Gene Expression; Humans; Nuclear Proteins; Obesity; Overnutrition; Poly A; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcription Factors
PubMed: 18608124
DOI: 10.1080/07853890802084878 -
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 -
The Journal of Biological Chemistry Aug 1992Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine,...
Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.
Topics: Amino Acids; Animals; Biological Transport; Female; Intestinal Mucosa; Jejunum; Kinetics; Oocytes; Poly A; RNA; RNA, Messenger; Rabbits; Sodium; Xenopus laevis
PubMed: 1379228
DOI: No ID Found -
Trends in Biochemical Sciences Jun 2013Cleavage and polyadenylation (C/P) of nascent transcripts is essential for maturation of the 3' ends of most eukaryotic mRNAs. Over the past three decades, biochemical... (Review)
Review
Cleavage and polyadenylation (C/P) of nascent transcripts is essential for maturation of the 3' ends of most eukaryotic mRNAs. Over the past three decades, biochemical studies have elucidated the machinery responsible for the seemingly simple C/P reaction. Recent genomic analyses have indicated that most eukaryotic genes have multiple cleavage and polyadenylation sites (pAs), leading to transcript isoforms with different coding potentials and/or variable 3' untranslated regions (UTRs). As such, alternative cleavage and polyadenylation (APA) is an important layer of gene regulation impacting mRNA metabolism. Here, we review our current understanding of APA and recent progress in this field.
Topics: Hydrolysis; Poly A; Proteome; Transcriptome
PubMed: 23632313
DOI: 10.1016/j.tibs.2013.03.005