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.     

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 roles of intersubunit interactions in exosome stability

In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species. A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface. So far, however, this model lacks experimental support. Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity. In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function. Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins.     

Analysis of the Saccharomyces cerevisiae exosome architecture and of the RNA binding activity of Rrp40p

The exosome is a complex of eleven subunits in yeast, involved in RNA processing and degradation. Despite the extensive in vivo functional studies of the exosome, little information is yet available on the structure of the complex and on the RNase and RNA binding activities of the individual subunits. The current model for the exosome structure predicts the formation of a heterohexameric RNase PH ring, bound on one side by RNA binding subunits, and on the opposite side by hydrolytic RNase subunits. Here, we report protein-protein interactions within the exosome, confirming the predictions of constituents of the RNase PH ring, and show some possible interaction interfaces between the other subunits. We also show evidence that Rrp40p can bind RNA in vitro, as predicted by sequence analysis.     

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 organization of the RNA-degrading exosome

The RNA exosome participates in the degradation and processing of a wide range of RNA molecules. Recent advances in understanding how the exosome is organized and functions largely stem from structural studies. Crystal structures of archaeal exosomes bound to RNA and of the corresponding nine-subunit human exosome core show that the archaeal and eukaryotic complexes have a similar molecular architecture, but have a diverged catalytic mechanism. The crystal structures of two hydrolytic RNases that associate with the exosome provide the framework for their catalytic activity. Negative-stain EM reconstructions give us a first glimpse of how they associate with the core complex. Together, these structural studies have implications for the mechanism of RNA recruitment and degradation by the exosome complexes.     

An exosome-like complex in Sulfolobus solfataricus

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cep1 synthetic lethality 4; an RNA-binding protein and exosome subunit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.     

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.     

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.     

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.     

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.     

The Bacillus subtilis RNA Helicase YxiN is Distended in Solution

The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.     

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.     

Elucidating the Role of Microprocessor Protein DGCR8 in Bending RNA Structures

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.     

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.     

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.     

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.