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.     

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 exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA.

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast

The biogenesis of a number of RNA species in eukaryotic cells requires 3' processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3' to 5' exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3' exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.     

Functions of the exosome in rRNA, snoRNA and snRNA synthesis.

The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.     

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.     

Rat1p and Rai1p function with the nuclear exosome in the processing and degradation of rRNA precursors

Exoribonucleases function in the processing and degradation of a variety of RNAs in all organisms. These enzymes play a particularly important role in the maturation of rRNAs and in a quality-control pathway that degrades rRNA precursors upon inhibition of ribosome biogenesis. Strains with defects in 3'-5' exoribonucleolytic components of the RNA processing exosome accumulate polyadenylated precursor rRNAs that also arise in strains with ribosome biogenesis defects. These findings suggested that polyadenylation might target pre-rRNAs for degradation by the exosome. Here we report experiments that indicate a role for the 5'-3' exoribonuclease Rat1p and its associated protein Rai1p in the degradation of poly(A)(+) pre-rRNAs. Depletion of Rat1p enhances the amount of poly(A)(+) pre-rRNA that accumulates in strains deleted for the exosome subunit Rrp6p and decreases their 5' heterogeneity. Deletion of RAI1 results in the accumulation of poly(A)(+) pre-rRNAs, and inhibits Rat1p-dependent 5'-end processing and Rrp6p-dependent 3'-end processing of 5.8S rRNA. RAT1 and RAI1 mutations cause synergistic growth defects in the presence of rrp6-Delta, consistent with the interdependence of 5'-end and 3'-end processing pathways. These findings suggest that Rai1p may coordinate the 5'-end and 3'-end processing and degradation activities of Rat1p and the nuclear exosome.     

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.     

Ngl2p is a Ccr4p-like RNA nuclease essential for the final step in 3'-end processing of 5.8S rRNA in Saccharomyces cerevisiae

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg(2+)-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2delta) demonstrates that correct 3'-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3'-extended 5.8S rRNA formed in the ngl2delta mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3' --> 5' exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3'-end formation of yeast 5.8S rRNA.     

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 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.     

The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Eukaryotes have two types of ribosomes containing either 5.8SL or 5.8SS rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8SL rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8SS rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8SL rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Delta3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Delta3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8SS and 5.8SL rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed.     

C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing

The exosome is a complex of 3'-5' exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein-protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3'-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3' end processing of the 5.8S rRNA.     

Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits

Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A(0) to A(2) and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A(3) and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp5Delta6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in the rrp5Delta6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A(2). Instead, the 32S precursor is processed directly at site A(3), producing exclusively 21S rather than 20S pre-rRNA. This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis. Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation. The resulting D-A(3) fragment, however, is degraded by both 5'-->3' and 3'-->5' exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5'-->3' exonucleolytic digestion of the D-A(2) fragment. We also show that rrp5Delta6 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal.     

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.     

The evolutionarily conserved protein Las1 is required for pre-rRNA processing at both ends of ITS2

Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3' end and for Rat1-dependent formation of the 25S rRNA 5' end. We further show that the Rat1-Rai1 5'-3' exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ~7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5' end of pre-25S RNAs suggestive of a protected spacer fragment of similar length.     

Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3'' end formation of 5.8S rRNA in Saccharomyces cerevisiae.

The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p). Dob1p is an essential putative ATP-dependent RNA helicase. Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant. Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs. Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor. This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p. In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA. A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition. We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.     

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.