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.     

Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA–DNA, DNA–RNA, and RNA–RNA duplexes with a long 3′ overhang (≥10 nucleotides). The C‐terminal tail (CTT)‐truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo‐form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase–Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell‐shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.     

Emerging themes in non-coding RNA quality control

Quality control pathways for non-coding RNAs such as tRNAs and rRNAs are widespread. In both prokaryotes and eukaryotes, poly(A) polymerases target aberrant non-coding RNAs for degradation. In yeast, a nuclear complex that includes the poly(A) polymerase Trf4p works together with the exosome in degrading a broad array of non-coding RNAs, several of which are aberrant. Yeast also have additional pathways for the degradation of defective RNAs and other pathways may exist in higher eukaryotes. One possibility is that cells recognize specific, still undiscovered, features common to misfolded RNAs; however, an alternative is that RNA quality control proteins interact with relatively general RNA structures, whereas correctly folded RNAs are sequestered by specific RNA-binding proteins and thus protected from degradation. Recently available structures of protein and ribonucleoprotein complexes involved in non-coding RNA quality control are providing a more detailed understanding of this process.     

RNA polyadenylation and degradation in different Archaea; roles of the exosome and RNase R

Polyadenylation is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most mRNAs. Contrarily, polyadenylation can stimulate RNA degradation, a phenomenon witnessed in prokaryotes, organelles and recently, for nucleus-encoded RNA as well. Polyadenylation takes place in hyperthermophilic archaea and is mediated by the archaeal exosome, but no RNA polyadenylation was detected in halophiles. Here, we analyzed polyadenylation in the third archaea group, the methanogens, in which some members contain genes encoding the exosome but others lack these genes. Polyadenylation was found in the methanogen, Methanopyrus kandleri, containing the exosome genes, but not in members which lack these genes. To explore how RNA is degraded in the absence of the exosome and without polyadenylation, we searched for the exoribonuclease that is involved in this process. No homologous proteins for any other known exoribonuclease were detected in this group. However, the halophilic archaea contain a gene homologous to the exoribonuclease RNase R. This ribonuclease is not able to degrade structured RNA better than PNPase. RNase R, which appears to be the only exoribonucleases in Haloferax volcanii, was found to be essential for viability.     

Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring

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.     

The exosome: a macromolecular cage for controlled RNA degradation

The exosome, a large multisubunit complex with exoribonucleic activity, emerges as the central 3′ RNA degradation and processing factor in eukaryotes and archaea. But how are the many RNA substrates of the exosome degraded in a processive, yet controlled manner? Recent functional and structural progress shows that the exosome is a macromolecular cage, where the nuclease active sites are situated in a central processing chamber. A narrow entry pore controls access to the active sites in the processing chamber and prevents uncontrolled RNA decay. The emerging mechanism of exosome function suggests a strikingly parallel architectural concept to protein degradation by proteasomes.     

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.     

Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance

RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a nine-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs. Loss of these regulatory functions results in specific metabolic and plasmid inheritance defects. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.     

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30–60 °C (pH 7.0), and at pH 4.0–7.0 (40 °C) for 10–14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3′-, 5′- and 2′-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 °C (pH 7.0). Autolysis at lower temperatures (30 °C and 40 °C) and at pH 5.0 and 6.0 favoured the formation of 3′-nucleotides, whereas autolysis at 40 °C and 50 °C (pH 7.0) favoured 5′- and 2′-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5′-AMP and 5′-GMP, were 50 °C (pH 7.0) and pH 4.0 (40 °C), respectively.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.     

Domain analysis of the chloroplast polynucleotide phosphorylase reveals discrete functions in RNA degradation,polyadenylation, and sequence homology with exosome proteins

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. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an alpha-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.     

RNA-quality control by the exosome

The exosome complex of 3'-->5' exonucleases is an important component of the RNA-processing machinery in eukaryotes. This complex functions in the accurate processing of nuclear RNA precursors and in the degradation of RNAs in both the nucleus and the cytoplasm. However, it has been unclear how different classes of substrate are distinguished from one another. Recent studies now provide insights into the regulation and structure of the exosome, and they reveal striking similarities between the process of RNA degradation in bacteria and eukaryotes.     

RNA polyadenylation in Archaea: not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.     

Running rings around RNA: a superfamily of phosphate-dependent RNases.

The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA. Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH. These enzymes are phosphorylases that degrade RNA from the 3'-end. A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily. Here, information from the structure of PNPase is used to address two related issues. First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme. Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction.     

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.