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.     

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.     

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.     

The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro

The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.     

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.     

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.     

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.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.     

Modulating the RNA Processing and Decay by the Exosome: Altering Rrp44/Dis3 Activity and End-Product

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.     

An RNA-Binding Complex Involved in Ribosome Biogenesis Contains a Protein with Homology to tRNA CCA-Adding Enzyme

A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA–protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.     

Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.     

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.     

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.     

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.     

Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.