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Query: EC:3.1.13.1 (exoribonuclease)
 732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPgammaS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3'-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3' termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.
J Mol Biol 1998 Jun 26
PMID:Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes. 964 84

Rap1p localization factor 4 (RLF4) is a Saccharomyces cerevisiae gene that was identified in a screen for mutants that affect telomere function and alter the localization of the telomere binding protein Rap1p. In rlf4 mutants, telomeric silencing is reduced and telomere DNA tracts are shorter, indicating that RLF4 is required for both the establishment and/or maintenance of telomeric chromatin and for the control of telomere length. In this paper, we demonstrate that RLF4 is allelic to NMD2/UPF2, a gene required for the nonsense-mediated mRNA decay (NMD) pathway (Y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Mol. Cell. Biol. 9:423-436, 1995, and F. He and A. Jacobson, Genes Dev. 9:437-454, 1995). The NMD pathway, which requires Nmd2p/Rlf4p together with two other proteins, (Upf1p and Upf3p), targets nonsense messages for degradation in the cytoplasm by the exoribonuclease Xrn1p. Deletion of UPF1 and UPF3 caused telomere-associated defects like those caused by rlf4 mutations, implying that the NMD pathway, rather than an NMD-independent function of Nmd2p/Rlf4p, is required for telomere functions. In addition, telomere length regulation required Xrn1p but not Rat1p, a nuclear exoribonuclease with functional similarity to Xrn1p (A. W. Johnson, Mol. Cell. Biol. 17:6122-6130, 1997). In contrast, telomere-associated defects were not observed in pan2, pan3, or pan2 pan3 strains, which are defective in the intrinsic deadenylation-dependent decay of normal (as opposed to nonsense) mRNAs. Thus, loss of the NMD pathway specifically causes defects at telomeres, demonstrating a physiological requirement for the NMD pathway in normal cell functions. We propose a model in which the NMD pathway regulates the levels of specific mRNAs that are important for telomere functions.
Mol Cell Biol 1998 Oct
PMID:Telomere length regulation and telomeric chromatin require the nonsense-mediated mRNA decay pathway. 974 29

The yeast nuclear gene DSS1 codes for a mitochondrial protein containing regions of homology to bacterial RNase II and can act as a multicopy suppressor of a deletion of the SUV3 gene, which encodes an RNA helicase. In order to establish the function of the DSS1 gene in mitochondrial biogenesis we studied RNA metabolism in yeast strains disrupted for SUV3 or DSS1. The results indicate that in the absence of DSS1 the in vitro activity of 3'-5' exoribonuclease is abolished and mitochondrial translation is blocked. In disruption strains harboring intronless mitochondrial genomes steady-state levels of COB mRNA and 16S rRNA were very low, while in the presence of a mitochondrial genome containing the omega intron in the 21S rRNA gene the excised intron accumulates. Moreover we observed an accumulation of precursors of 21S rRNA and the VAR1 mRNA. All these phenotypes are virtually identical to those of strains in which SUV3 is disrupted. We suggest that the DSS1 gene product, like the SUV3 gene product, is a subunit of the yeast mitochondrial degradosome (mtEXO), and that this protein complex participates in intron-independent turnover and processing of mitochondrial transcripts. In addition our studies exclude any role for the NUC1 nuclease in these phenomena.
Mol Gen Genet 1998 Oct
PMID:The yeast nuclear gene DSS1, which codes for a putative RNase II, is necessary for the function of the mitochondrial degradosome in processing and turnover of RNA. 982 34

Metabolic instability is a hallmark property of mRNAs in most if not all organisms and plays an essential role in facilitating rapid responses to regulatory cues. This article provides a critical examination of recent progress in the enzymology of mRNA decay in Escherichia coli, focusing on six major enzymes: RNase III, RNase E, polynucleotide phosphorylase, RNase II, poly(A) polymerase(s), and RNA helicase(s). The first major advance in our thinking about mechanisms of RNA decay has been catalyzed by the possibility that mRNA decay is orchestrated by a multicomponent mRNA-protein complex (the "degradosome"). The ramifications of this discovery are discussed and developed into mRNA decay models that integrate the properties of the ribonucleases and their associated proteins, the role of RNA structure in determining the susceptibility of an RNA to decay, and some of the known kinetic features of mRNA decay. These models propose that mRNA decay is a vectorial process initiated primarily at or near the 5' terminus of susceptible mRNAs and propagated by successive endonucleolytic cleavages catalyzed by RNase E in the degradosome. It seems likely that the degradosome can be tethered to its substrate, either physically or kinetically through a preference for monphosphorylated RNAs, accounting for the usual "all or none" nature of mRNA decay. A second recent advance in our thinking about mRNA decay is the rediscovery of polyadenylated mRNA in bacteria. Models are provided to account for the role of polyadenylation in facilitating the 3' exonucleolytic degradation of structured RNAs. Finally, we have reviewed the documented properties of several well-studied paradigms for mRNA decay in E. coli. We interpret the published data in light of our models and the properties of the degradosome. It seems likely that the study of mRNA decay is about to enter a phase in which research will focus on the structural basis for recognition of cleavage sites, on catalytic mechanisms, and on regulation of mRNA decay.
Prog Nucleic Acid Res Mol Biol 1999
PMID:Degradation of mRNA in Escherichia coli: an old problem with some new twists. 993 52

A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.
Mol Cell Biol 1999 Mar
PMID:NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae. 1002 25

The rpsO mRNA of E. coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I. We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E. It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II. A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2. Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells. Poly(A)polymerase I contributes differently to the instability of these fragments. The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript. We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA.
J Mol Biol 1999 Mar 05
PMID:E. coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent degradation of the mRNA. 1004 80

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
Mol Microbiol 1999 Dec
PMID:Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. 1059 33

The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5. 8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.
Mol Cell Biol 2000 Jun
PMID:Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p. 1080 43

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single-stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3' termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.
Mol Microbiol 2000 May
PMID:Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. 1084 84

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.
Mol Microbiol 2001 Mar
PMID:RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II. 1126 Apr 72


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