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.     

Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.     

Crystal Structure of the S. solfataricus Archaeal Exosome Reveals Conformational Flexibility in the RNA-Binding Ring

Background

The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/Principal Findings

Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/Significance

This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.     

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 and biochemical characterization of the yeast exosome component Rrp40

The exosome is a protein complex that is important in both degradation and 3'-processing of eukaryotic RNAs. We present the crystal structure of the Rrp40 exosome subunit from Saccharomyces cerevisiae at a resolution of 2.2 A. The structure comprises an S1 domain and an unusual KH (K homology) domain. Close packing of the S1 and KH domains is stabilized by a GxNG sequence, which is uniquely conserved in exosome KH domains. Nuclear magnetic resonance data reveal the presence of a manganese-binding site at the interface of the two domains. Isothermal titration calorimetry shows that Rrp40 and archaeal Rrp4 alone have very low intrinsic affinity for RNA. The affinity of an archaeal core exosome for RNA is significantly increased in the presence of the S1-KH subunit Rrp4, indicating that multiple subunits might contribute to cooperative binding of RNA substrates by the exosome.     

A single subunit, Dis3, is essentially responsible for yeast exosome core activity

The conserved core of the exosome, the major eukaryotic 3' --> 5' exonuclease, contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3' --> 5' exoRNases. We show here that Dis3 is responsible for exosome core activity. The purified exosome core has a hydrolytic, processive and Mg(2+)-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically not different from wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function.     

The Pyrococcus exosome complex: structural and functional characterization

The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.     

Dis3‐like 1: a novel exoribonuclease associated with the human exosome

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.     

Poring over exosome structure

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.164Almost all RNA molecules are processed by RNases to form mature RNAs. In addition, many RNAs are degraded, either because they are no longer needed or because they are aberrant. All of these functions—RNA processing, normal RNA degradation and RNA quality control—are carried out by the eukaryotic RNA exosome complex. In this issue of EMBO reports, the Lorentzen group provide structural insight into the eukaryotic exosome and the mechanism by which it degrades RNA from 3′ to 5′ (Malet et al, 2010).The crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006; Bonneau et al, 2009) and the related bacterial PNPase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these RNA-degrading machines from the three domains of life have a similar structure (Fig 1). They are all composed of a ring of six RNase PH domains, one side of which has a cap that contains putative RNA-binding domains. Although this overall structure is conserved, the way that it is formed is not. Bacterial PNPase is a homotrimer of which each monomer contains two RNase PH domains, an S1 domain and a KH domain. The archaeal PH ring consists of three copies of two proteins and the cap is made of three copies of either one of two proteins. Finally, the eukaryotic exosome core is composed of nine proteins: six with one RNase PH domain each and three cap proteins.Open in a separate windowFigure 1Exosome structures. The bacterial PNPase (left), the archaeal exosome (middle) and eukaryotic core exosome (right) have a common overall structure. The top panels are schematic views from above, showing the cap proteins. The bottom panels show a view from the side, with one-third of the exosome cut away to reveal the RNA in the central channel.In PNPase and the archaeal exosome, substrates enter the PH ring from the cap-side. The putative RNA-binding domains of the cap are therefore probably important for controlling entry to the PH ring. In both archaea and bacteria, the active sites are on the inner side of the PH ring and thus the ribonucleic catalysis occurs inside the central channel. However, in humans and yeast each of the RNase PH domains have point mutations that make the exosome ring catalytically inactive (Dziembowski et al, 2007). Instead, catalysis is carried out by a tenth subunit—Rrp44/Dis3—which binds to the PH ring on the opposite side to the cap proteins (Bonneau et al, 2009; Wang et al, 2007). This organization made it unclear whether RNA also enters the central channel of the exosome in eukaryotes (Fig 1), or whether substrate RNAs directly access the catalytic subunit.Malet and colleagues now provide structural information that resolves this by reconstituting the ten-subunit yeast exosome and analysing its structure with electron microscopy, in the presence and absence of RNA. This analysis suggests that the RNase PH ring of the exosome is stable, but that the cap and catalytic subunits are more flexible than previously appreciated. It is the first structural evidence that in eukaryotes RNA is threaded through the central channel before being degraded by Rrp44.     

Structural framework for the mechanism of archaeal exosomes in RNA processing

Exosomes emerge as central 3'-->5' RNA processing and degradation machineries in eukaryotes and archaea. We determined crystal structures of two 230 kDa nine subunit archaeal exosome isoforms. Both exosome isoforms contain a hexameric ring of RNase phosphorolytic (PH) domain subunits with a central chamber. Tungstate soaks identified three phosphorolytic active sites in this processing chamber. A trimer of Csl4 or Rrp4 subunits forms a multidomain macromolecular interaction surface on the RNase-PH domain ring with central S1 domains and peripheral KH and zinc-ribbon domains. Structural and mutational analyses suggest that the S1 domains and a subsequent neck in the RNase-PH domain ring form an RNA entry pore to the processing chamber that only allows access of unstructured RNA. This structural framework can mechanistically unify observed features of exosomes, including processive degradation of unstructured RNA, the requirement for regulatory factors to degrade structured RNA, and left-over tails in rRNA trimming.     

Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core

The exosome is a macromolecular complex that plays fundamental roles in the biogenesis and turnover of a large number of RNA species. Here we report the crystal structures of the Rrp41-Rrp42 core complex of the S. solfataricus exosome bound to short single-stranded RNAs and to ADP. The RNA binding cleft recognizes four nucleotides in a sequence-unspecific manner, mainly by electrostatic interactions with the phosphate groups. Interactions at the 2' hydroxyls of the sugars provide specificity for RNA over DNA. The structures show both the bound substrate and the cleaved product of the reaction, suggesting a catalytic mechanism for the 3'-5' phosphorolytic activity of the exosome.     

Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

Ribonuclease P (RNase P) catalyzes the removal of 5′ leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.     

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 decay machines: The exosome

The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell.The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate.The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3′-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p–core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome.     

Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family

The eukaryotic exosome is a macromolecular complex essential for RNA processing and decay. It has recently been shown that the RNase activity of the yeast exosome core can be mapped to a single subunit, Rrp44, which processively degrades single-stranded RNAs as well as RNAs containing secondary structures. Here we present the 2.3 A resolution crystal structure of S. cerevisiae Rrp44 in complex with single-stranded RNA. Although Rrp44 has a linear domain organization similar to bacterial RNase II, in three dimensions the domains have a different arrangement. The three domains of the classical nucleic-acid-binding OB fold are positioned on the catalytic domain such that the RNA-binding path observed in RNase II is occluded. Instead, RNA is threaded to the catalytic site via an alternative route suggesting a mechanism for RNA-duplex unwinding. The structure provides a molecular rationale for the observed biochemical properties of the RNase R family of nucleases.     

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.