Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.
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PMID:Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach. 1613 13

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.
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PMID:In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence. 1765 49

In this article, we report on the genetic analysis of the Schizosaccharomyces pombe open reading frames SPCC1322.01 and SPAC637.11, respectively, which encode proteins that are similar to the exoribonuclease Dss1p and the RNA helicase Suv3p, respectively, forming the mitochondrial degradosome of Saccharomyces cerevisiae. While the helicase Suv3p is exchangeable between S. cerevisiae and S. pombe, the functions of Dss1p and the putative fission yeast RNase protein are specific for each species. Unlike S. cerevisiae mutants lacking a functional degradosome, the major defect of fission yeast knock-out strains is their inability to perform downstream processing of transcripts. In addition, the lack of pah1 results in instability of mitochondrial RNA ends. Overexpression of par1 and pah1 has no significant effect on the steady-state levels of mitochondrial RNAs. The Pet127p-stimulated RNA degradation activity is independent of Par1p/Pah1p in fission yeast mitochondria. The results presented herein indicate that both fission yeast proteins play only a minor role (if at all) in mitochondrial RNA degradation. We assume that the RNA-degrading function was taken over by other enzymes in fission yeast mitochondria, while the former degradosome proteins were recruited to new cellular pathways, for example, RNA processing in fission yeast (as discussed in this article) or mitochondrial DNA replication, apoptosis, or chromatin maintenance in eukaryotes, during evolution.
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PMID:The 3' ends of mature transcripts are generated by a processosome complex in fission yeast mitochondria. 1830 78

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli, it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components: the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. The variable presence of additional proteins, however, suggests that the degradosome is a flexible machine that may vary its composition in response to different conditions. Direct analysis of large protein complexes, together with simplified purification procedures, can facilitate qualitative and quantitative identification of RNA degradosome components under different physiologic and genetic conditions and can help to explain their role in the bacterial cell (see also Chapters 4, 11, 19, 20 and 22 regarding methods for the studying the degradosome and other multiprotein complexes in this volume. Herewith we describe the application of multidimensional protein identification technology (MudPIT) in the rapid and quantitative identification of RNA degradosome components. RNA degradosome preparations obtained from specific conditions are enzymatically digested. The resulting peptides are fractionated using two-dimensional (ion-exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. Bioinformatic analysis with the SEQUEST algorithm, which correlates experimentally obtained mass spectra with those predicted from peptide sequences in proteomic and translated genomic databases, allows identification of the corresponding proteins that compose the complex. The protein constituents of two or more degradosome samples are then compared to obtain a rapid evaluation of qualitative and quantitative differences in protein composition. Quantitative analysis is based on the observation that changes in relative protein abundance among different samples are reflected by statistical parameters (score values) assigned to each protein component of the RNA degradosome identified by the MudPIT approach. This correlation can be validated by independent methods such as Western blotting and determination of enzymatic activities. This fully automated procedure may be applied to the characterization of any complex protein mixture.
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PMID:A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli. 1916 40

The mitochondrial degradosome (mtEXO) of S. cerevisiae is the main exoribonuclease of yeast mitochondria. It is involved in many pathways of mitochondrial RNA metabolism, including RNA degradation, surveillance, and processing, and its activity is essential for mitochondrial gene function. The mitochondrial degradosome is a very simple example of a 3' to 5'-exoribonucleolytic complex. It is composed of only two subunits: Dss1p, which is an RNR (RNase II-like) family exoribonuclease, and Suv3p, which is a DExH/D-box RNA helicase. The two subunits form a tight complex and their activities are highly interdependent, with the RNase activity greatly enhanced in the presence of the helicase subunit, and the helicase activity entirely dependent on the presence of the ribonuclease subunit. In this chapter, we present methods for studying the function of the yeast mitochondrial degradosome in vivo, through the analysis of degradosome-deficient mutant yeast strains, and in vitro, through heterologous expression in E. coli and purification of the degradosome subunits and reconstitution of a functional complex. We provide the protocols for studying ribonuclease, ATPase, and helicase activities and for measuring the RNA binding capacity of the complex and its subunits.
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PMID:In vivo and in vitro approaches for studying the yeast mitochondrial RNA degradosome complex. 1916 56

Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H(2)O(2)) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H(2)O(2), PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required.
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PMID:Polynucleotide phosphorylase protects Escherichia coli against oxidative stress. 1921 92

Mitochondrial RNA turnover in yeast involves the degradosome, composed of DSS-1 exoribonuclease and SUV3 RNA helicase. Here, we describe a degradosome-like complex, containing SUV3 and DSS-1 homologues, in the early branching protozoan, Trypanosoma brucei. TbSUV3 is mitochondrially localized and co-sediments with TbDSS-1 on glycerol gradients. Co-immunoprecipitation demonstrates that TbSUV3 and TbDSS-1 associate in a stable complex, which differs from the yeast degradosome in that it is not stably associated with mitochondrial ribosomes. This is the first report of a mitochondrial degradosome-like complex outside of yeast. Our data indicate an early evolutionary origin for the mitochondrial SUV3/DSS-1 containing complex.
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PMID:Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei. 1954 Feb 36

Endoribonuclease E, 3'-5' exoribonuclease polynucleotide phosphorylase, RhlB RNA helicase and enolase form a multienzyme complex (the "degradosome") playing an important role in RNA processing and decay in Escherichia coli. Although a number of proteins that occasionally co-purify with the E. coli degradosome in non-stoichiometric amounts were initially viewed as insignificant constituents of this complex, recent studies suggest that, in fact, some of these copurifying proteins are involved in modulation of degradosome composition, activity or specificity during bacterial adaptation to changing environments. Here we briefly review these findings and discuss their implications for understanding the multifaceted mechanisms controlling degradosome functions in vivo.
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PMID:Unraveling new roles for minor components of the E. coli RNA degradosome. 1966 55

Here we report a unique processing pathway in Escherichia coli for tRNA(Leu5) in which the exoribonuclease polynucleotide phosphorylase (PNPase) removes the Rho-independent transcription terminator from the leuX transcript without requiring the RhlB RNA helicase. Our data demonstrate for the first time that PNPase can efficiently degrade an RNA substrate containing secondary structures in vivo. Furthermore, RNase P, an endoribonuclease that normally generates the mature 5'-ends of tRNAs, removes the leuX terminator inefficiently independent of PNPase activity. RNase P cleaves 4-7 nt downstream of the CCA determinant generating a substrate for RNase II, which removes an additional 3-4 nt. Subsequently, RNase T completes the 3' maturation process by removing the remaining 1-3 nt downstream of the CCA determinant. RNase E, G and Z are not involved in terminator removal. These results provide further evidence that the E. coli tRNA processing machinery is far more diverse than previously envisioned.
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PMID:Processing of the Escherichia coli leuX tRNA transcript, encoding tRNA(Leu5), requires either the 3'-->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator. 1990 95

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.
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PMID:The critical role of RNA processing and degradation in the control of gene expression. 2067 45


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