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1.
Properties of the exonuclease activity that degrades H4 histone mRNA   总被引:28,自引:0,他引:28  
We have described a cell-free system for studying mRNA degradation (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Using that system we found that human H4 histone mRNA was degraded in a 3' to 5' direction by an exonucleolytic activity. Here we investigate several properties of the crude system and of the exonuclease. A RNase inhibitor, such as that from placenta, was required to block nonspecific ribonucleases and thereby to permit different mRNAs to be degraded at different rates. The histone mRNA exonuclease required divalent cation (magnesium) but not exogenously added ATP or GTP. It functioned efficiently at monovalent cation concentrations ranging from 0.5 to 200 mM. It was bound to ribosomes isolated from cells lysed in low salt buffers. However, it was eluted in active form from the ribosomes by exposing them to 0.3 M KCl. The enzyme rapidly degraded deproteinized, 32P-labeled histone mRNA prepared enzymatically.  相似文献   

2.
Circular RNAs are abundant,conserved, and associated with ALU repeats   总被引:10,自引:0,他引:10  
Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.  相似文献   

3.
The relative decay of four human messenger RNAs, gamma globin, delta globin, c-myc and H4 histone, were compared in a cell-free system. Under appropriate conditions, they are degraded in vitro in approximately the same relative order as in vivo: histone faster than c-myc and delta globin faster than gamma globin. Degradation of polysome-associated H4 histone mRNA and of deproteinized histone mRNA begins at or near the 3' terminus. At least a portion of the mRNA then continues to be degraded in a 3' to 5' direction. Discrete 3'-terminal degradation hold-up points are observed, suggesting that 3' to 5' degradation occurs non-uniformly. Cycloheximide and puromycin inhibit protein synthesis but do not affect the rate or directionality of histone mRNA decay in vitro. We conclude that the rate-limiting step in H4 histone mRNA decay occurs at or near the 3' terminus and that at least a portion of the mRNA molecule is subsequently degraded 3' to 5', probably via a processive exonuclease.  相似文献   

4.
5.
The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions.  相似文献   

6.
Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently. These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared. Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage. The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules. Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.  相似文献   

7.
The degradation of ribonucleic acids injected into Xenopus laevis oocytes   总被引:14,自引:0,他引:14  
C C Allende  J E Allende  R A Firtel 《Cell》1974,2(3):189-196
Different radioactive RNAs were injected into Xenopus laevis oocytes, and their degradation followed with time. Deproteinized ribosomal RNAs and synthetic polynucleotides, with the exception of polyadenylic acid, were degraded rapidly with apparent first order kinetics and half-lives ranging from 1–6 hr. Transfer RNA, poly(A), and ribosomal RNA injected as whole ribosomal particles were quite stable during the period studied (20 hr). Messenger RNAs from Dictyostelium discoideum and Vesicular Stomatitis Virus, which have poly(A) sequences at their 3′ terminus, presented biphasic degradation kinetics. Approximately 60% of these RNAs was degraded in the first 6 hr, whereas the remaining 30–40% was stable for at least 22 hr. Analysis of the stable material by sucrose gradients showed that it had the same sedimentation pattern as the original material, except that it contained, in addition, free poly(A) sequences sedimenting somewhat smaller than 4S. Puromycin treatment of the cells injected with Dictyostelium mRNAs reduced the percentage of stable RNA to 10%, approximately the poly(A) content of these RNAs. Similar treatment with emetine, which also inhibited cellular protein synthesis, did not affect the stable mRNA fraction.  相似文献   

8.
The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.  相似文献   

9.
10.
The stem–loop binding protein (SLBP) binds the 3′ end of histone mRNA and is present both in nucleus, and in the cytoplasm on the polyribosomes. SLBP participates in the processing of the histone pre-mRNA and in translation of the mature message. Histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication and are stabilized by inhibitors of translation, resulting in an increase in histone mRNA levels. Here, we show that SLBP is a component of the histone messenger ribonucleoprotein particle (mRNP). Histone mRNA from polyribosomes is immunoprecipitated with anti-SLBP. Most of the SLBP in cycloheximide-treated cells is present on polyribosomes as a result of continued synthesis and transport of the histone mRNP to the cytoplasm. When cells are treated with inhibitors of DNA replication, histone mRNAs are rapidly degraded but SLBP levels remain constant and SLBP is relocalized to the nucleus. SLBP remains active both in RNA binding and histone pre-mRNA processing when DNA replication is inhibited.  相似文献   

11.
Low molecular weight RNA species from chromatin.   总被引:4,自引:0,他引:4  
Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.  相似文献   

12.
The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.  相似文献   

13.
Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T1 ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T1 oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.  相似文献   

14.
Kempf BJ  Barton DJ 《Journal of virology》2008,82(12):5835-5846
Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.  相似文献   

15.
16.
Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.  相似文献   

17.
18.
These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.  相似文献   

19.
The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.  相似文献   

20.
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