Structure and regulation of the nuclear exosome targeting complex guides RNA substrates to the exosome

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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.     

Quantitative analysis of processive RNA degradation by the archaeal RNA exosome

RNA exosomes are large multisubunit assemblies involved in controlled RNA processing. The archaeal exosome possesses a heterohexameric processing chamber with three RNase-PH-like active sites, capped by Rrp4- or Csl4-type subunits containing RNA-binding domains. RNA degradation by RNA exosomes has not been studied in a quantitative manner because of the complex kinetics involved, and exosome features contributing to efficient RNA degradation remain unclear. Here we derive a quantitative kinetic model for degradation of a model substrate by the archaeal exosome. Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates. We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed. Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain. We also show that formation of the RNase-PH like ring with entrapped RNA is not required for high catalytic efficiency, suggesting that the exosome chamber evolved for controlled processivity, rather than for catalytic chemistry in RNA decay.     

Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex

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MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts

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Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.     

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.     

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.     

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.     

The oligomeric architecture of the archaeal exosome is important for processive and efficient RNA degradation

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3′ end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events.     

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 channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.     

RNA channelling by the archaeal exosome

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.     

Degradation of ribosomal RNA precursors by the exosome

The yeast exosome is a complex of 3′→5′ exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3′ end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A₀, A₁, A₂ and A₃, reducing the levels of the 32S, 20S, 27SA₂ and 27SA₃ pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3′→5′ exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans-acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A₂-C₂ RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.     

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.     

Contribution of domain structure to the RNA 3' end processing and degradation functions of the nuclear exosome subunit Rrp6p

The 3'-5' riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3' ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3' ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3'-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.