Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.     

mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures

Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3′–5′ exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3′–5′ exonuclease PNPase at stem-loop structures in vitro. This factor can, potentialiy, reconcile the apparent discrepancy between the ability of 3′ stem-loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.     

Nuclear EGFR Suppresses Ribonuclease Activity of Polynucleotide Phosphorylase through DNAPK-mediated Phosphorylation at Serine 776

Nuclear existence of epidermal growth factor receptor (EGFR) has been documented for more than two decades. Resistance of cancer to radiotherapy is frequently correlated with elevated EGFR expression, activity, and nuclear translocation. However, the role of nuclear EGFR (nEGFR) in radioresistance of cancers remains elusive. In the current study, we identified a novel nEGFR-associated protein, polynucleotide phosphorylase (PNPase), which possesses 3' to 5' exoribonuclease activity toward c-MYC mRNA. Knockdown of PNPase increased radioresistance. Inactivation or knock-down of EGFR enhanced PNPase-mediated c-MYC mRNA degradation in breast cancer cells, and also increased its radiosensitivity. Interestingly, the association of nEGFR with PNPase and DNA-dependent protein kinase (DNAPK) increased significantly in breast cancer cells after exposure to ionizing radiation (IR). We also demonstrated that DNAPK phosphorylates PNPase at Ser-776, which is critical for its ribonuclease activity. The phospho-mimetic S776D mutant of PNPase impaired its ribonuclease activity whereas the nonphosphorylatable S776A mutant effectively degraded c-MYC mRNA. Here, we uncovered a novel role of nEGFR in radioresistance, and that is, upon ionizing radiation, nEGFR inactivates the ribonuclease activity of PNPase toward c-MYC mRNA through DNAPK-mediated Ser-776 phosphorylation, leading to increase of c-MYC mRNA, which contributes to radioresistance of cancer cells.     

Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP. As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.     

The PNPase,exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

The structure and function of polynucleotide phosphorylase (PNPase) and the exosome, as well as their associated RNA-helicases proteins, are described in the light of recent studies. The picture raised is of an evolutionarily conserved RNA-degradation machine which exonucleolytically degrades RNA from 3′ to 5′. In prokaryotes and in eukaryotic organelles, a trimeric complex of PNPase forms a circular doughnut-shaped structure, in which the phosphorolysis catalytic sites are buried inside the barrel-shaped complex, while the RNA binding domains create a pore where RNA enters, reminiscent of the protein degrading complex, the proteasome. In some archaea and in the eukaryotes, several different proteins form a similar circle-shaped complex, the exosome, that is responsible for 3′ to 5′ exonucleolytic degradation of RNA as part of the processing, quality control, and general RNA degradation process. Both PNPase in prokaryotes and the exosome in eukaryotes are found in association with protein complexes that notably include RNA helicase.     

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.     

Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli

The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.     

Replication initiator protein mRNA of ColE2 plasmid and its antisense regulator RNA are under the control of different degradation pathways

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5' untranslated region. We showed that the Rep mRNA is subjected to complex degradation pathways involving at least RNase I, RNase II, RNase III, RNase E, RNase G and PNPase. RNase II acts as a major exoribonuclease and PNPase plays a minor role. We also showed that the PcnB (polyA polymerase I) plays only a minor role in the Rep mRNA degradation process. The RNA degradation pathways of the Rep mRNA and RNAI of the ColE2 plasmid are quite different. Based on these results, we speculate that the ColE2 Rep mRNA and RNAI are endowed with individual RNA half lives required for the efficient copy number control by being subjected to different RNA degradation systems.     

The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers

In Escherichia coli, REP-stabilizers are structural elements in polycistronic messages that protect 5'-proximal cistrons from 3'-->5' exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP-stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP-stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD-box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP-stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP-stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.     

Endonucleolytic activation directs dark-induced chloroplast mRNA degradation

Plastid mRNA stability is tightly regulated by external signals such as light. We have investigated the biochemical mechanism responsible for the dark-induced decrease of relative half-lives for mRNAs encoding photosynthetic proteins. Protein fractions isolated from plastids of light-grown and dark-adapted plants correctly reproduced an RNA degradation pathway in the dark that is downregulated in the light. This dark-dependent pathway is initiated by endonucleolytic cleavages in the petD mRNA precursor substrate proximal to a region that can fold into a stem–loop structure. Polynucleotide phosphorylase (PNPase) polyadenylation activity was strongly increased in the protein fraction isolated from plastids in dark-adapted plants, but interestingly PNPase activity was not required for the initiation of dark-induced mRNA degradation. A protein factor present in the protein fraction from plastids of light-grown plants could inactivate the endonuclease activity and thereby stabilize the RNA substrate in the protein fraction from plastids of dark-adapted plants. The results show that plastid mRNA stability is effectively controlled by the regulation of a specific dark-induced RNA degradation pathway.     

Nucleic acid and protein factors involved in Escherichia coli polynucleotide phosphorylase function on RNA

It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3′ end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5′ end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 °C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseΔS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.     

Analysis of the human polynucleotide phosphorylase (PNPase) reveals differences in RNA binding and response to phosphate compared to its bacterial and chloroplast counterparts

PNPase is a major exoribonuclease that plays an important role in the degradation, processing, and polyadenylation of RNA in prokaryotes and organelles. This phosphorolytic processive enzyme uses inorganic phosphate and nucleotide diphosphate for degradation and polymerization activities, respectively. Its structure and activities are similar to the archaeal exosome complex. The human PNPase was recently localized to the intermembrane space (IMS) of the mitochondria, and is, therefore, most likely not directly involved in RNA metabolism, unlike in bacteria and other organelles. In this work, the degradation, polymerization, and RNA-binding properties of the human PNPase were analyzed and compared to its bacterial and organellar counterparts. Phosphorolytic activity was displayed at lower optimum concentrations of inorganic phosphate. Also, the RNA-binding properties to ribohomopolymers varied significantly from those of its bacterial and organellar enzymes. The purified enzyme did not preferentially bind RNA harboring a poly(A) tail at the 3' end, compared to a molecule lacking this tail. Several site-directed mutations at conserved amino acid positions either eliminated or modified degradation/polymerization activity in different manners than observed for the Escherichia coli PNPase and the archaeal and human exosomes. In light of these results, a possible function of the human PNPase in the mitochondrial IMS is discussed.     

Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation

Bacterial polynucleotide phosphorylase (PNPase) plays a major role in mRNA turnover by the degradation of RNA from the 3′- to 5′-ends. Here, we determined the crystal structures of the wild-type and a C-terminal KH/S1 domain-truncated mutant (ΔKH/S1) of Escherichia coli PNPase at resolutions of 2.6 Å and 2.8 Å, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The truncated mutant ΔKH/S1 bound and cleaved RNA less efficiently with an eightfold reduced binding affinity. Thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that ΔKH/S1 formed a less stable trimer than the full-length PNPase. The crystal structure of ΔKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically. Moreover, site-directed mutagenesis of several arginine residues in the channel neck regions produced defective PNPases that either bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, indicating that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.     

The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes

In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.