Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in <i>Escherichia coli</i>

Briani, Federica; Curti, Serena Maria; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

doi:10.1261/rna.1123908

Cited by 42 publications

(65 citation statements)

References 76 publications

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“…In the absence of RNase III that pathway would be inoperative, or would operate at a reduced level, and the level of the rpsO transcript would increase. A second possibility relates to the observation that PNPase itself has been shown to stabilize transcripts in E. coli (5,23), including the rpsO transcript (5). While the exact mechanism of this stabilization is unknown, it is conceivable that the increased levels of PNPase in the absence of RNase III result in the stabilization of the rpsO transcript, and thus its increase, in S. coelicolor.…”

Section: Discussionmentioning

confidence: 99%

RNase III-Dependent Expression of the rpsO-pnp Operon of Streptomyces coelicolor

2011

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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 IIIdependent 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.Polynucleotide phosphorylase (PNPase) is a 3Ј-5Ј-exoribonuclease that functions in the phosphorolytic degradation of RNA molecules in bacteria and in eukaryotic organelles (14,21). In Escherichia coli and other bacteria, PNPase plays an important role in the degradation of mRNAs. Thus, endonucleolytic cleavage of RNA molecules generates 3Ј ends that are substrates for the action of PNPase and RNase II, an exonuclease that functions hydrolytically (8,12,28). PNPase plays another role in E. coli, at least under some circumstances. As is the case in eukaryotes, the 3Ј ends of at least some RNA molecules in bacteria are polyadenylated (31, 32). Polyadenylation facilitates the degradation of RNAs in bacteria (22,24,27). While the major enzyme responsible for RNA polyadenylation in E. coli is poly(A) polymerase I (PAP I [7]), mutants of E. coli lacking PAP I still retain the ability to polyadenylate RNAs (26), indicating that there is at least one other polyadenylating enzyme in those cells. Mohanty and Kushner have presented evidence indicating that the second PAP in E. coli is none other than PNPase (25). They argue that under appropriate conditions in vivo, PNPase can serve to degrade RNAs or synthesize poly(A) tails and that this enzyme is responsible for the G, C, and U residues that are found at low frequency in the poly(A) tails of RNAs from wild-type E. coli (25).PNPase structure, function, and expression have been studied extensively in species of the soil-dwelling, antibiotic-pro...

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Section: Discussionmentioning

confidence: 99%

RNase III-Dependent Expression of the rpsO-pnp Operon of Streptomyces coelicolor

2011

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“…To test whether this residual RNase III-independent autogenous regulation required phosphorolytic, RNA binding, or both PNPase activities, we measured posttranscriptional repression levels by PNPase mutants affected in either activity, namely, Pnp-⌬KHS1, which is missing the two RNA binding domains (31,33), and Pnp-S438A (with a mutation of S to A at position 438), which is devoid of phosphorolytic activity (25). To test whether the mutant retained RNA binding activity, we performed RNA-PNPase cross-linking experiments, as previously described (28). As shown by the results in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The PNPase content of the samples was evaluated by Western blot analysis of 400-ng samples of total protein using anti-PNPase antibodies (28) and densitometric analysis of the film using ImageQuant software (Molecular Dynamics). Electrophoretic mobility shift assays (EMSAs) were performed as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

“…1) for 20 min at 21°C in binding buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM DTT, 0.025% NP-40 [Fluka], and 10% glycerol) with either 400 ng of crude extract or purified proteins in a final volume of 10 l. The samples were UV irradiated (254-nm wavelength at 2.8 J/cm 2 ) and treated with RNase A, and the cross-linked proteins were fractionated by 10% SDS-PAGE and analyzed by phosphorimaging (28). RNA probes were obtained by T7 RNA polymerase transcription of DNA templates produced by PCR with the primers listed in Table 2 and plasmid pAZ101 as a template.…”

Section: Methodsmentioning

confidence: 99%

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RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

2015

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The complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5= end by RNase III, rather than the primary pnp transcript (RNase III-dependent models), and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic ⌬pnp rnc ؉ and ⌬pnp ⌬rnc strains with a chromosomal pnp-lacZ translational fusion and measured ␤-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1. IMPORTANCEIn Escherichia coli, polynucleotide phosphorylase (PNPase, encoded by pnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of pnp mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.A wealth of mechanisms that control gene expression and an intricate network of regulatory interactions subtly and promptly adapt the presence and concentration of gene products to a variety of environmental and developmental conditions. Autogenous regulation of the pnp gene in Escherichia coli has long since been considered an example of regulation at the level of mRNA stability. This gene codes for polynucleotide phosphorylase (PNPase), a phosphorolytic 3=-to-5= exoribonuclease and a template-independent, nucleoside diphosphate-dependent RNA polymerase that is conserved in bacteria and eukaryotic organelles (1, 2). E. coli PNPase plays a major role in RNA turnover and metabolism (3) and has been implicated in several processes, such as adaptation and growth in the cold, biofilm formation, and responses to oxidative stress...

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“…Oligonucleotides PL101/21 and PL102/19 were used for 16S rRNA reverse transcription and PCR amplification. mRNA half-lives were estimated as described [36] by regression analysis of mRNA remaining (estimated by real time PCR) versus time after rifampicin addition. Cell aggregation in C-1a (pnp + ), C-5691 (Δpnp) and C-5691 derivatives carrying mutations in genes encoding for adhesion determinants (ΔpgaC, C-5937; ΔbcsA, C-5929; ΔcsgA, C-5931; ΔwcaD, C-5935).…”

Section: Gene Expression Determinationmentioning

confidence: 99%

The RNA Processing Enzyme Polynucleotide Phosphorylase Negatively Controls Biofilm Formation by Repressing Poly-N-Acetylglucosamine (PNAG) Production in Escherichia coli C

Carzaniga¹,

Antoniani²,

Dehò³

et al. 2014

Biofilm Control and Antimicrobial Agents

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Background: Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode. Results: Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E. coli strain C-1a, in which deletion of the pnp gene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG), thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. Indeed, pgaABCD transcript levels are higher in the pnp mutant. Negative control of pgaABCD expression by PNPase takes place at mRNA stability level and involves the 5'-untranslated region of the pgaABCD transcript, which serves as a cis-element regulating pgaABCD transcript stability and translatability.

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Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli

Cited by 42 publications

References 76 publications

RNase III-Dependent Expression of the rpsO-pnp Operon of Streptomyces coelicolor

RNase III-Dependent Expression of the rpsO-pnp Operon of Streptomyces coelicolor

RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

The RNA Processing Enzyme Polynucleotide Phosphorylase Negatively Controls Biofilm Formation by Repressing Poly-N-Acetylglucosamine (PNAG) Production in Escherichia coli C

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