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.     

The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.     

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.     

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 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.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Assembly of the Yeast Exoribonuclease Rrp6 with Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47

Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.     

The exosome‐binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase

The exosome is a conserved multi‐subunit ribonuclease complex that functions in 3′ end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10‐subunit core complex of the exosome (Exo‐10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo‐10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N‐terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N‐terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C‐terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6–Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.     

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.     

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.     

Nab3 Facilitates the Function of the TRAMP Complex in RNA Processing via Recruitment of Rrp6 Independent of Nrd1

Non-coding RNAs (ncRNAs) play critical roles in gene regulation. In eukaryotic cells, ncRNAs are processed and/or degraded by the nuclear exosome, a ribonuclease complex containing catalytic subunits Dis3 and Rrp6. The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is a critical exosome cofactor in budding yeast that stimulates the exosome to process/degrade ncRNAs and human TRAMP components have recently been identified. Importantly, mutations in exosome and exosome cofactor genes cause neurodegenerative disease. How the TRAMP complex interacts with other exosome cofactors to orchestrate regulation of the exosome is an open question. To identify novel interactions of the TRAMP exosome cofactor, we performed a high copy suppressor screen of a thermosensitive air1/2 TRAMP mutant. Here, we report that the Nab3 RNA-binding protein of the Nrd1-Nab3-Sen1 (NNS) complex is a potent suppressor of TRAMP mutants. Unlike Nab3, Nrd1 and Sen1 do not suppress TRAMP mutants and Nrd1 binding is not required for Nab3-mediated suppression of TRAMP suggesting an independent role for Nab3. Critically, Nab3 decreases ncRNA levels in TRAMP mutants, Nab3-mediated suppression of air1/2 cells requires the nuclear exosome component, Rrp6, and Nab3 directly binds Rrp6. We extend this analysis to identify a human RNA binding protein, RALY, which shares identity with Nab3 and can suppress TRAMP mutants. These results suggest that Nab3 facilitates TRAMP function by recruiting Rrp6 to ncRNAs for processing/degradation independent of Nrd1. The data raise the intriguing possibility that Nab3 and Nrd1 can function independently to recruit Rrp6 to ncRNA targets, providing combinatorial flexibility in RNA processing.     

Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells

Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.     

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.     

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.