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Plant mitochondrial polyadenylated mRNAs are degraded by a 3'- to 5'-exoribonuclease activity, which proceeds unimpeded by stable secondary structures.

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3'-extremities. We show that a 3'- to 5'-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20-30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3' to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3' ends of mRNAs promote an efficient 3'- to 5'- degradation process.

Base Sequence↗

A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease.

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A greater than p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme-substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.

Adenosine↗

[Isolation and some properties of exoribonuclease from the cell nuclei of the rat liver].

Exoribonuclease from rat liver cell nuclei was isolated and purified using selective extraction at pH 8.0, salting out with ammonium sulfate (30-50% saturation) and chromatography on DEAE-Sephadex A-50. The enzyme has the pH optimum 7.4-7.6, it requires Mg-2+. It catalyses the gradual removal of ribonucleoside-5'-monophosphates from synthetic polynucleotides and RNA; it preferably attacks single-stranded polynucleotides and is less efficient when hydrolysing poly-stranded helical polymers. It is supposed that exoribonuclease can participate in the destruction of non-informative regions in new-formed hyper-molecular RNA of cell nuclei.

Animals↗

Influence of the xyloadenosine analogue of 2',5'-oligoriboadenylate on poly(A)-specific, 2',5'-oligoriboadenylate degrading 2',3'-exoribonuclease and further enzymes involved in poly(A)(+)mRNA metabolism.

The homogeneous poly(A)-specific 2',3'-exoribonuclease from calf thymus gland, which cleaves both 3',5'- and 2',5'-linked oligoriboadenylates, does not degrade (xyloA2'p)2 xyloA, the xylofuranosyladenosine analogue of the 2-5A core. This oligonucleotide, which is supposed to enter intact cells rapidly, was found to possess an increased stability and an enhanced antiherpesvirus activity compared to the natural (A2'p)2A (Eppstein, D.A., Barnett, J.W., Marsh, Y.V., Gosselin, G. and Imbach, J.-L. (1983) Nature 302, 723-724). The poly(A) anabolic enzyme, poly(A) polymerase (Mn2+-dependent), from the same source, which is initiated by (A3'p)2A and its higher oligomers, does not accept 2-5A core and its xyloadenosine analogue as primer. Both oligonucleotides exert no influence on endoribonuclease IV and on the integrity of the poly(A)-ribonucleoprotein complex.

Adenine Nucleotides↗

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity.

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).

Exoribonucleases↗

Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth.

As a step toward determining the metabolic role(s) of a 5'----3' exoribonuclease (XRN1), a yeast gene, XRN1, encoding XRN1, was first cloned, then disrupted to test its essentially or effect on yeast cell growth. Clones in the high-copy-number plasmid YEp24 cause overproduction (fivefold) of XRN1 in yeast cells, as measured by either poly(A) hydrolytic activity or immunoreactivity. Restriction mapping and deletion analysis showed that the XRN1 gene is located on a 6.7-kb XbaI-XhoI fragment of chromosome VII. The normal gene was disrupted in two haploid yeast strains by integrating a fragment with a BglII-deleted segment replaced with the yeast URA3 gene, and the disrupted strains lack XRN1. Successful transformation of haploid cells showed that the gene is not essential, but its absence markedly affected the cell growth rate. The growth defect is corrected by introduction of the XRN1 gene on a plasmid back into the disrupted yeast.

Blotting, Southern↗

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.

Exoribonucleases↗

The TCL1 oncoprotein binds the RNase PH domains of the PNPase exoribonuclease without affecting its RNA degrading activity.

TCL1 is an AKT kinase coactivator that, when dysregulated, initiates mature lymphocyte malignancies in humans and transgenic mice. While TCL1 augments AKT pathway signaling, additional TCL1 interacting proteins that may contribute to cellular homeostasis or transformation are lacking. Here, an exoribonuclease, PNPase, was identified in a complex with TCL1. The AKT interaction domain on TCL1 bound either RNase PH repeat domain of PNPase without influencing its RNA degrading activity, which was compatible with predicted docking models for a TCL1-PNPase complex. Our data provide a novel protein interaction for mammalian PNPase that may impact TCL1 mediated transformation.

Blotting, Western↗

The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity.

The yeast mitochondrial protein Suv3p is a putative NTP-dependent RNA helicase. Here we report that in cells lacking Suv3p, there is an approximately 50-fold increase in the excised form of the group I intron omega of the mitochondrial 31S rRNA gene. Surprisingly, little mature 21S rRNA accumulates in those cells; instead, unligated 21S rRNA exons appear. Intron overaccumulation could lead to spliced exon reopening via a reaction known to be catalyzed by group I introns in vitro. We also show that Suv3p is a functional component of a novel mitochondrial NTP-dependent 3'-to-5' exoribonuclease activity that can degrade group I intron RNAs. These findings account for group I intron overaccumulation in cells lacking Suv3p and define a novel function for putative RNA helicases in direct RNA degradation.

Base Sequence↗

In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA.

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.

Alleles↗

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis.

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.

Amino Acid Sequence↗

ETHYLENE-INSENSITIVE5 encodes a 5'-->3' exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2.

Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence of ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F-box proteins, EBF1 and EBF2. Here we report the identification of ETHYLENE-INSENSITIVE5 as the 5'-->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response.

Alleles↗

A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 3'-->5' exoribonuclease activity.

The processing of precursor tRNAs at their 5' and 3' termini is a fundamental event in the biosynthesis of tRNA. RNase P is generally responsible for endonucleolytic removal of a leader sequence of precursor tRNA to generate the mature 5' terminus. However, much less is known about the RNase P counterparts or other proteins that are active at the tRNA 3' terminus. Here we show that one of the human RNase P subunits, Rpp14, together with one of its interacting protein partners, OIP2, is a 3'-->5' exoribonuclease with a phosphorolytic activity that processes the 3' terminus of precursor tRNA. Immunoprecipitates of a crude human RNase P complex can process both ends of precursor tRNA by hydrolysis, but purified RNase P has no exonuclease activity. Rpp14 and OIP2 may be part of an exosome activity.

Carrier Proteins↗

Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease.

3'-Exonucleolytic removal of the poly(A) tail is the first and often rate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplasmic extract from HeLa cells, the poly(A) tail of mRNA was degraded from the 3'-end. In agreement with earlier in vivo observations, prominent decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for this poly(A) shortening activity was purified from calf thymus. A polypeptide of 74 kDa copurified with the activity. The deadenylating nuclease (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly dependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly(A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. At either salt concentration DAN and PAB I reconstituted poly(A) shortening with the same pattern of intermediates seen in cytoplasmic extract. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.

Animals↗

The mechanism of preferential degradation of polyadenylated RNA in the chloroplast. The exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence.

Polyadenylation of mRNA in the chloroplast has recently been shown to target the RNA molecule for rapid exonucleolytic degradation. A model has been suggested in which the degradation of chloroplast mRNA is initiated by endonucleolytic cleavage(s) followed by the addition of poly(A)-rich sequences and rapid exonucleolytic degradation. When in vitro transcribed RNAs were incubated with chloroplast protein extract, competition between polyadenylated and non-polyadenylated RNAs for degradation resulted in the rapid degradation of the polyadenylated molecules and stabilization of their non-polyadenylated counterparts. To elucidate the molecular mechanism governing this effect, we determined whether the chloroplast exoribonuclease 100RNP/polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated 100RNP/PNPase degraded polyadenylated and non-polyadenylated RNAs at the same rate. However, when both molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. In RNA binding experiments, 100RNP/PNPase bound the poly(A) sequence with much higher affinity than other RNA molecules, thereby defining the poly(A)-rich RNA as a preferential substrate for the enzyme. 100RNP/PNPase may therefore be involved in a mechanism in which post-transcriptional addition of poly(A)-rich sequence targets the chloroplast RNA for rapid exonucleolytic degradation.

Binding Sites↗

The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R.

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3'-5' exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.

Bacterial Proteins↗

Purification and characterization of the Escherichia coli exoribonuclease RNase R. Comparison with RNase II.

Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.

Chromatography, Gel↗

A 5'----3' exoribonuclease of human placental nuclei: purification and substrate specificity.

An exoribonuclease that hydrolyzes single-stranded RNA by a 5'----3' mode yielding 5'-mononucleotides has been purified from human placental nuclei. Chromatographic studies of crude placental nuclear extracts suggest that the enzyme is a relatively abundant nuclear RNase. Poly(A) is degraded by a processive mechanism while rRNA is degraded in a partially non-processive manner, possibly because of its secondary structure. The enzyme has an apparent molecular weight of 113,000, derived from determinations of the Stokes radius (43 A) and sedimentation coefficient (6.3 S). Substrates with 5'-phosphomonoester end groups are 10-20 times better than 5'-dephosphorylated substrates. The locale of the enzyme in nuclei of normal human cells as well as its mode of action suggest a role in nuclear RNA processing or turnover.

Cell Nucleus↗