Crystal structure of the phosphorolytic exoribonuclease RNase PH from Bacillus subtilis and implications for its quaternary structure and tRNA binding

RNase PH is a member of the family of phosphorolytic 3' --> 5' exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X-ray diffraction to medium resolution. Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively. The structures contain a rare left-handed beta alpha beta-motif in the N-terminal portion of the protein. This motif has also been identified in other enzymes involved in RNA metabolism. The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N-terminal core of the structure and active site of Streptomyces antibioticus PNPase. The surface of the RNase PH dimer fit the shape of a tRNA molecule.     

Novel role for RNase PH in the degradation of structured RNA

Escherichia coli contains multiple 3' to 5' RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3' end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.     

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.     

Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain

In the present work we have used a double-hybrid assay in bacteria to identify a putative domain in E. coli PNPase required for in vivo interaction with RNase E. We used a 202 aa fragment of RNase E previously reported as the PNPase binding domain in this enzyme and a collection of 13 different fragments of 105 aa, spanning the entire sequence of 734 aa PNPase (GenBank Accession number NP_417633). Our results indicate that two clones of PNPase including residues 158-262 and residues 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration. Three-dimensional modeling of the E. coli PNPase based on the S. antibioticus protein structure indicates that the putative binding domain is located on the monomer surface, facing outward from the trimeric tertiary structure. Since a copy of the betabetaalphabetabetaalpha domain is also found in RNase PH, we investigated and found an interaction with RNase E in a pull-down assay. We suggest this interaction takes place through the similar betabetaalphabetabetaalpha domain present in the tertiary structure of this enzyme. Based on these results, we propose that RNase PH and RNase E could form functional assemblies in E. coli.     

Characterization of Staphylococcus epidermidis Polynucleotide phosphorylase and its interactions with ribonucleases RNase J1 and RNase J2

Polynucleotide phosphorylase catalyzes both 3′-5′ exoribonuclease and polyadenylation reactions. The crystal structure of Staphylococcus epidermidis PNPase revealed a bound phosphate in the PH2 domain of each protomer coordinated by three adjacent serine residues. Mutational analysis suggests that phosphate coordination by these serine residues is essential to maintain the catalytic center in an active conformation. We note that PNPase forms a complex with RNase J1 and RNase J2 without substantially altering either exo-ribonuclease or polyadenylation activity of this enzyme. This decoupling of catalytic activity from protein-protein interactions suggests that association of these endo- or exo-ribonucleases with PNPase could be more relevant for cellular localization or concerted targeting of structured RNA for recycling.     

Degradation of ribosomal RNA during starvation: comparison to quality control during steady-state growth and a role for RNase PH

Ribosomal RNAs are generally stable in growing Escherichia coli cells. However, their degradation increases dramatically under conditions that lead to slow cell growth. In addition, incomplete RNA molecules and molecules with defects in processing, folding, or assembly are also eliminated in growing cells in a process termed quality control. Here, we show that there are significant differences between the pathways of ribosomal RNA degradation during glucose starvation and quality control during steady-state growth. In both processes, endonucleolytic cleavage of rRNA in ribosome subunits is an early step, resulting in accumulation of large rRNA fragments when the processive exoribonucleases, RNase II, RNase R, and PNPase are absent. For 23S rRNA, cleavage is in the region of helix 71, but the exact position can differ in the two degradative processes. For 16S rRNA, degradation during starvation begins with shortening of its 3' end in a reaction catalyzed by RNase PH. In the absence of this RNase, there is no 3' end trimming of 16S rRNA and no accumulation of rRNA fragments, and total RNA degradation is greatly reduced. In contrast, the degradation pattern in quality control remains unchanged when RNase PH is absent. During starvation, the exoribonucleases RNase II and RNase R are important for fragment removal, whereas for quality control, RNase R and PNPase are more important. These data highlight the similarities and differences between rRNA degradation during starvation and quality control during steady-state growth and describe a role for RNase PH in the starvation degradative pathway.     

The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization

RNase II is a 3'-5' exoribonuclease that processively hydrolyzes single-stranded RNA generating 5' mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 3'-5' exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.     

RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II

In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°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 Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain.     

Action of RNase II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure

The 3'-->5' exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3' extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.     

The origin of polynucleotide phosphorylase domains

In this report, we document the presence of polynucleotide phosphorylase (PNPase) in the animal eukaryotes. These proteins contain several domains, including 2 RNase PH domains (PNPase 1 and PNPase 2) which are closely related functionally and in sequence similarity to ribonuclease PH (RPH) protein. Phylogenetic analysis of the gene genealogy of these three domains suggests that PNPase was formed via a duplication event that also produced the RNase PH protein. Given the current distribution of these domains in the tree of life, these duplication events most likely occurred in the common ancestor of the three organismal superkingdoms, Archaea, Eukarya, and Bacteria. In particular, PNPase 2 and RPH are more closely related to each other than either one is to PNPase 1, suggesting a deeper differentiation of PNPase 1 in the common organismal ancestor. In addition, while PNPase 1 and PNPase 2 appear to have the same evolutionary signal as determined by the incongruence length difference (ILD) test, RPH appears to have an incongruent signal with both of the PNPase domains. This result suggests that RPH experienced different evolutionary divergence patterns than the PNPase domains, consistent with the linked nature of the two PNPase domains.     

Decay of the IS 10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn 10/IS 10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3’exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3′ exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens RNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only twofold across the normal range of physiological growth temperatures (30–44°C) in wild-type cells, which has important implications for IS 10 biology.     

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 archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3' --> 5' exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 A. The structure reveals a hexameric ring-like arrangement of three Rrp41-Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA-degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.     

Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel β-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.     

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 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.     

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.