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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human polynucleotide phosphorylase (hPNPase(old-35)) is a type I IFN-inducible 3',5' exoribonuclease that mediates mRNA degradation. In melanoma cells, slow and sustained overexpression of hPNPase(old-35) induces G(1) cell cycle arrest ultimately culminating in apoptosis, whereas rapid overexpression of hPNPase(old-35) directly promotes apoptosis without cell cycle changes. These observations imply that inhibition of cell cycle progression and induction of apoptosis by hPNPase(old-35) involve multiple intracellular targets and signaling pathways. We now provide evidence that the apoptosis-inducing activity of hPNPase(old-35) is mediated by activation of double-stranded RNA-dependent protein kinase (PKR). Activation of PKR by hPNPase(old-35) precedes phosphorylation of eukaryotic initiation factor-2alpha and induction of growth arrest and DNA damage-inducible gene 153 (GADD153) that culminates in the shutdown of protein synthesis and apoptosis. Activation of PKR by hPNPase(old-35) also instigates down-regulation of the antiapoptotic protein Bcl-x(L). A dominant-negative inhibitor of PKR, as well as GADD153 antisense or bcl-x(L) overexpression, effectively inhibits apoptosis induction by hPNPase(old-35). These studies elucidate a novel pathway by which an evolutionary conserved RNA-metabolizing enzyme, hPNPase(old-35), regulates cell growth and viability.
Cancer Res 2007 Sep 01
PMID:Activation of double-stranded RNA dependent protein kinase, a new pathway by which human polynucleotide phosphorylase (hPNPase(old-35)) induces apoptosis. 1780

In Escherichia coli, the cold shock response is exerted upon a temperature change from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3'-to-5' exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3'-to-5' exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3'-to-5' processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Delta pnp cells has consequences, such as accumulation of ribosomal subunits in the Delta pnp cells, which may play a role in the cold sensitivity of this strain.
J Bacteriol 2008 Sep
PMID:RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II. 1860 34

RNases are involved in critical aspects of RNA metabolism in all organisms. Two classes of RNases that digest RNA from an end (exo-RNases) are known: RNases that use water as a nucleophile to catalyze RNA degradation (hydrolytic RNases) and RNases that use inorganic phosphate (phosphorolytic RNases). It has been shown previously that the absence of the two known Escherichia coli phosphorolytic RNases, polynucleotide phosphorylase and RNase PH, leads to marked growth and ribosome assembly defects. To investigate the basis for these defects, a screen for growth suppressors was performed. The majority of suppressor mutations were found to lie within nsrR, which encodes a nitric oxide (NO)-sensitive transcriptional repressor. Further analysis showed that the suppressors function not by inactivating nsrR but by causing overexpression of a downstream gene that encodes a hydrolytic RNase, RNase R. Additional studies revealed that overexpression of another hydrolytic RNase, RNase II, similarly suppressed the growth defects. These results suggest that the requirement for phosphorolytic RNases for robust cellular growth and efficient ribosome assembly can be bypassed by increased expression of hydrolytic RNases.
J Bacteriol 2009 Sep
PMID:Identification and characterization of growth suppressors of Escherichia coli strains lacking phosphorolytic ribonucleases. 1961 68

In Escherichia coli, translational arrest can elicit cleavage of codons within the ribosomal A site. This A-site mRNA cleavage is independent of RelE, and has been proposed to be an endonucleolytic activity of the ribosome. Here, we show that the 3'-->5' exonuclease RNase II plays an important role in RelE-independent A-site cleavage. Instead of A-site cleavage, translational pausing in DeltaRNase II cells produces transcripts that are truncated +12 and +28 nucleotides downstream of the A-site codon. Deletions of the genes encoding polynucleotide phosphorylase (PNPase) and RNase R had little effect on A-site cleavage. However, PNPase overexpression restored A-site cleavage activity to DeltaRNase II cells. Purified RNase II and PNPase were both unable to directly catalyse A-site cleavage in vitro. Instead, these exonucleases degraded ribosome-bound mRNA to positions +18 and +24 nucleotides downstream of the ribosomal A site respectively. Finally, a stable structural barrier to exoribonuclease activity inhibited A-site cleavage when introduced immediately downstream of paused ribosomes. These results demonstrate that 3'-->5' exonuclease activity is an important prerequisite for efficient A-site cleavage. We propose that RNase II degrades mRNA to the downstream border of paused ribosomes, facilitating cleavage of the A-site codon by an unknown RNase.
Mol Microbiol 2009 Sep
PMID:RNase II is important for A-site mRNA cleavage during ribosome pausing. 1962 1

The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5' region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5' end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5' terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5' end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3'-proximal sites was an early step in turnover of mRNA.
J Bacteriol 2009 Sep
PMID:Processing and stability of inducibly expressed rpsO mRNA derivatives in Bacillus subtilis. 1963 85

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3' transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3'-to-5' exonuclease processing from the native 3' end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5' tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5' end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.
J Biol Chem 2009 Sep 25
PMID:Bacillus subtilis trp Leader RNA: RNase J1 endonuclease cleavage specificity and PNPase processing. 1963 40

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.
J Bacteriol 2010 Sep
PMID:(p)ppGpp inhibits polynucleotide phosphorylase from streptomyces but not from Escherichia coli and increases the stability of bulk mRNA in Streptomyces coelicolor. 2058 Dec 11

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.
FEMS Microbiol Rev 2010 Sep
PMID:The critical role of RNA processing and degradation in the control of gene expression. 2067 45

We have examined the expression of the rpsO-pnp operon in an RNase III (rnc) mutant of Streptomyces coelicolor. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the rnc mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that rpsO-pnp mRNA levels increased in the rnc mutant by 1.6- to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of rpsO in the rpsO-pnp operon; the pnp transcript, initiated in the rpsO-pnp intergenic region; and the rpsO transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the rpsO-pnp operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase III-dependent manner in S. coelicolor. To explore further the increase in the level of the pnp transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified S. coelicolor RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the pnp transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.
J Bacteriol 2011 Sep
PMID:RNase III-dependent expression of the rpsO-pnp operon of Streptomyces coelicolor. 2174 67

In human mitochondria, 10 mRNAs species are generated from a long polycistronic precursor that is transcribed from the heavy chain of mitochondrial DNA, in theory yielding equal copy numbers of mRNA molecules. However, the steady-state levels of these mRNAs differ substantially. Through absolute quantification of mRNAs in HeLa cells, we show that the copy numbers of all mitochondrial mRNA species range from 6000 to 51,000 molecules per cell, indicating that mitochondria actively regulate mRNA metabolism. In addition, the copy numbers of mitochondrial mRNAs correlated with their cellular half-life. Previously, mRNAs with longer half-lives were shown to be stabilized by the LRPPRC/SLIRP complex, which we find that cotranscriptionally binds to coding sequences of mRNAs. We observed that the LRPPRC/SLIRP complex suppressed 3' exonucleolytic mRNA degradation mediated by PNPase and SUV3. Moreover, LRPPRC promoted the polyadenylation of mRNAs mediated by mitochondrial poly(A) polymerase (MTPAP) in vitro. These findings provide a framework for understanding the molecular mechanism of mRNA metabolism in human mitochondria.
Nucleic Acids Res 2012 Sep
PMID:LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and promotes polyadenylation in human mitochondria. 2266 77


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