The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome

Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3′ exonuclease activity. We report that expression only of Rrp44 lacking 3′-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5′-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3′-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo–exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5′-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.     

Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3′→5′ exonuclease containing S1 and KH RNA-binding domains

The exosome, an evolutionarily conserved complex of multiple 3′→5′ exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3′→5′ exonuclease that requires a free 3′-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5′-monophosphate products. AtRrp4p behaves as an ~500 kDa species during sedimentation through a 10–30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.     

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.     

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.     

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.     

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.     

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.     

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.     

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.     

Reconstitution, activities, and structure of the eukaryotic RNA exosome

The RNA exosome is a multisubunit 3' to 5' exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.     

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.     

Genetic interactions suggest multiple distinct roles of the arch and core helicase domains of Mtr4 in Rrp6 and exosome function

The RNA exosome is responsible for a wide variety of RNA processing and degradation reactions. The activity and specificity of the RNA exosome is thought to be controlled by a number of cofactors. Mtr4 is an essential RNA-dependent adenosine triphosphatase that is required for all of the nuclear functions of the RNA exosome. The crystal structure of Mtr4 uncovered a domain that is conserved in the RNA exosome cofactors Mtr4 and Ski2 but not in other helicases, suggesting it has an important role related to exosome activation. Rrp6 provides the nuclear exosome with one of its three nuclease activities, and previous findings suggested that the arch domain is specifically required for Rrp6 functions. Here, we report that the genetic interactions between the arch domain of Mtr4 and Rrp6 cannot be explained by the arch domain solely acting in Rrp6-dependent processing reactions. Specifically, we show that the arch domain is not required for all Rrp6 functions, and that the arch domain also functions independently of Rrp6. Finally, we show that the arch domain of Ski2, the cytoplasmic counterpart of Mtr4, is required for Ski2’s function, thereby confirming that the arch domains of these cofactors function independently of Rrp6.     

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.     

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.     

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.     

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.