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

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.
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PMID:RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli. 1094 97

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.
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PMID:Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli. 1132 69

In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S(1) mapping we defined the 3'-end at 8423(+/-1); thus CI RNA is 79(+/-1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5'-end and the CCA sequence at the 3'-end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.
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PMID:Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity. 1181 28

mRNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript. The RNase e of Escherichia coli is a single-strand-specific endo-nuclease involved in the processing of rRNA and the degradation of mRNA. A nucleolytic multi-enzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E. Two other components are a 3' exoribonuclease (polynucleotide phosphorylase, PNPase) and a DEAD-box RNA helicase (RNA helicase B, RhlB). RNase E is a large multidomain protein with N-terminal ribonucleolytic activity, an RNA-binding domain and a C-terminal "scaffold" that binds PNPase, enolase and RhlB. RhlB by itself has little activity but is strongly stimiulated by its interaction with RNase E. RhlB in vitro can facilitate the degradation of structured RNA by PNPase. Since the discovery of the RNA degradosome in E. coli, related complexes have been described in other organisms.
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PMID:The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes. 1203 60

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is a multifunctional protein, with a 3'-5' processive exoribonuclease, a Pi exchange, an RNA polymerase and an autoregulatory activity. The interaction between this enzyme and the mRNA target is crucial for its activities. In the present study, we characterized the interaction of PNPase with its mRNA regulatory region and ssRNA, as well as with ssDNA and dsDNA by determining K(d). Our results indicate that PNPase has high affinity for its mRNA, ssRNA and for ssDNA (K(d) approximately 10-20 nM). However, this enzyme exhibits a lower affinity for dsDNA (K(d) approximately 200-1400 nM). Possible implications of these results on the molecular mechanisms by which PNPase is regulated and degrades mRNA are discussed.
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PMID:Polynucleotide phosphorylase binds to ssRNA with same affinity as to ssDNA. 1210 10

RNase E contains a large non-catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non-catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a P(T7)-lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non-catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under P(lac) control. In contrast to rne+,low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non-catalytic part are necessary for normal activity in vivo.
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PMID:Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA. 1220 92

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. Here we present a model for the assembly of the six human RNase PH-like exosome subunits into a hexameric ring structure. In part, this structure is on the basis of the evolutionarily related bacterial degradosome, the core of which consists of three copies of the PNPase protein, each containing two RNase PH domains. In our model three additional exosome subunits, which contain S1 RNA-binding domains, are positioned on the outer surface of this ring. Evidence for this model was obtained by the identification of protein-protein interactions between individual exosome subunits in a mammalian two-hybrid system. In addition, the results of co-immunoprecipitation assays indicate that at least two copies of hRrp4p and hRrp41p are associated with a single exosome, suggesting that at least two of these ring structures are present in this complex. Finally, the identification of a human gene encoding the putative human counterpart of the bacterial PNPase protein is described, which suggests that the exosome is not the eukaryotic equivalent of the bacterial degradosome, although they do share similar functional activities.
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PMID:Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring. 1241 56

An overlapping pathway screening (OPS) approach designed to identify and clone genes displaying parallel expression profiles as a function of induction of terminal differentiation and cellular senescence in human cells identified a novel gene old-35. Sequence and functional analysis indicates that old-35 encodes human polynucleotide phosphorylase, hPNPase(old-35). Polynucleotide phosphorylases comprise a family of phosphate dependent 3'-5' RNA exonucleases implicated in RNA regulation. Treatment of HO-1 human melanoma and additional diverse normal and tumor-derived human cell types with Type I interferon (IFN), IFN-beta or IFN-alpha, induces hPNPase(old-35) expression. To provide insights into the regulation of hPNPase(old-35), we cloned and analyzed the promoter region of this gene. These studies demonstrate that IFN-beta controls hPNPase(old-35) expression by transcriptional modulation rather than by altering mRNA stability. Transcriptional activation of hPNPase(old-35) by IFN-beta is primarily mediated by the interferon stimulatory response element (ISRE) present in its promoter. Analysis of hPNPase(old-35) expression in cell lines defective in various IFN signaling molecules confirms that hPNPase(old-35) expression is dependent upon the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Furthermore, gel shift analyses document that hPNPase(old-35) is a direct target of the interferon stimulated gene factor 3 (ISGF3) complex. The hPNPase(old-35) gene spans approximately 54 kb of genomic DNA and is distributed on 28 exons and 27 introns. hPNPase(old-35) maps to 2p15-2p16.1, a region implicated in hereditary nonpolyposis colorectal cancer, Carney complex, Doyne's honeycomb retinal dystrophy and several other diseases. To provide insights into PNPase function in vivo, we have also cloned the mouse PNPase(old-35) cDNA, mPNPase(old-35). Induction of hPNPase(old-35) by IFN treatment as well as during differentiation and senescence suggest that this gene may play a significant role in regulating cellular growth and that overlapping gene expression changes, also induced by IFN, may contribute to these important physiological processes.
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PMID:Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPase(old-35), a Type I interferon inducible early response gene. 1456 61

In this paper we show that RNase R is a cold shock protein that is induced seven- to eightfold by cold shock and that its expression is tightly regulated by temperature. Transcriptional studies reveal that the rnr gene is co-transcribed with flanking genes as an operon induced under cold shock. The induction of RNase R levels is mainly a result of the stabilization of the rnr transcripts. The transient stability of the rnr transcripts is shown to be regulated by PNPase at the end of the acclimation phase. Studies with an rnr mutant revealed a cold-shock phenotype showing that RNase R contributes to growth at low temperatures. We have shown that RNase R can be involved in the maturation of SsrA/tmRNA, an important small stable RNA involved in protein tagging and ribosome rescue. The wide biological significance of RNase R regarding adaptation to cold shock and its involvement in RNA surveillance, protein quality control and pathogenesis is discussed.
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PMID:Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA. 1462 21

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.
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PMID:The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation. 1465 5


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