Rrp44 Harbors Endonuclease Activity (Nucleic Acids Research 2009) PDF

Published online 7 January 2009 Nucleic Acids Research, 2009, Vol. 37, No. 4 1127–1140 doi:10.1093/nar/gkn1020 The N-terminal PIN domain of t...

Published online 7 January 2009 Nucleic Acids Research, 2009, Vol. 37, No. 4 1127–1140 doi:10.1093/nar/gkn1020 The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome Claudia Schneider1, Eileen Leung2, Jeremy Brown2 and David Tollervey1,* 1 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR and 2RNA Biology Group and Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK Received November 14, 2008; Revised December 4, 2008; Accepted December 7, 2008 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 ABSTRACT components are shared between the nuclear and cytoplas- mic forms of the complex, and all of these are essential for Nuclear and cytoplasmic forms of the yeast exo- cell viability (1). The nuclear exosome participates in some share 10 components, of which only Rrp44/ many RNA degradation and surveillance pathways, as Dis3 is believed to possess 3’ exonuclease activity. well as processing the precursors to the 5.8S rRNA and We report that expression only of Rrp44 lacking other stable RNA species (2). The cytoplasmic exosome 3’-exonuclease activity (Rrp44-exo) supports functions in mRNA degradation, participating in general growth in S288c-related strains (BY4741). In mRNA turnover and several activated decay and surveil- BY4741, rrp44-exo was synthetic-lethal with loss of lance pathways (3). the cytoplasmic 5’-exonuclease Xrn1, indicating Structural and functional analyses indicate that Rrp44 block of mRNA turnover, but not with loss of the (Dis3) is the only catalytically active 30 –50 exonuclease in nuclear 3’-exonuclease Rrp6. The RNA processing the yeast exosome core (4–6), whereas the nuclear exo- some is associated with a second active nuclease (Rrp6) phenotype of rrp44-exo was milder than that seen (7). Rrp44 is related to Escherichia coli RNase R, a on Rrp44 depletion, indicating that Rrp44-exo member of the RNase II (RNase B) family of hydrolytic retains important functions. Recombinant Rrp44 exonucleases. As shown in Figure 1A, Rrp44 has an was shown to possess manganese-dependent N-terminal PIN-domain (PilT N-terminus—derived from endonuclease activity in vitro that was abolished the name of an E. coli protein implicated in pilus forma- by four point mutations in the putative metal binding tion), which is not shared with RNase R or RNase II. residues of its N-terminal PIN domain. Rrp44 lacking Recent studies reported endonuclease activity associated both exonuclease and endonuclease activity failed with the PIN-domains of human Smg6 and yeast Swt1, to support growth in strains depleted of endoge- both of which are implicated in mRNA surveillance (8,9), nous Rrp44. Strains expressing Rrp44-exo and and the PIN domain protein Nob1 was predicted to be a Rrp44-endo–exo exhibited different RNA proces- pre-rRNA endonuclease (10,11). The PIN domain in sing patterns in vivo suggesting Rrp44-dependent Rrp44 is followed by a putative RNA-binding, cold- shock domain (CSD), for which functional analyses have endonucleolytic cleavages in the 5’-ETS and ITS2 not yet been reported. The exonuclease activity resides in regions of the pre-rRNA. Finally, the N-terminal the RNB domain and this is abolished by the point muta- PIN domain was shown to be necessary and suffi- tion D551N (4,6). A C-terminal S1 RNA-binding domain cient for association with the core exosome, indi- is also important for substrate binding and for activity cating its dual function as a nuclease and in vitro and in vivo, and this is impaired by the G916E structural element. mutation (6,12). Strains lacking the exonuclease activity of Rrp44 are viable, whereas the integrity of the exosome complex is essential since depletion of any single core component is INTRODUCTION lethal (1,4–6). These observations raised several questions, The exosome complex is implicated in many RNA proces- including whether other nucleases in the nucleus or cyto- sing and degradation activities. Ten ‘core’ exosome plasm can functionally substitute for the exonuclease *To whom correspondence should be addressed. Tel: + 44 131 650 7092; Fax: +44 131 650 7040; Email: [email protected] ß 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1128 Nucleic Acids Research, 2009, Vol. 37, No. 4 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 Figure 1. In vivo analysis of yeast strains expressing Rrp44-exo. (A) Domain structures of E. coli RNase II and S. cerevisiae Rrp44. Positions of Rrp44 point mutations are indicated. (B) Growth analysis of yeast strains expressing Rrp44-exo. A GAL::rrp44 yeast strain transformed with plasmids encoding Rrp44 [WT or D551N (exo)] or the empty vector pRS316 were grown in liquid selective glucose medium at 308C to analyze growth. (C) Structure of the 35S pre-rRNA with the location of oligonucleotide probes used for Northern hybridization. (D) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with a plasmid expressing either WT Rrp44 or the mutant (D551N) Rrp44-exo protein, or an empty vector. RNA was isolated from GAL::rrp44 strains grown at 308C under permissive conditions (GAL) and 8 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and either detected by Northern hybridization with the oligonucleo- tide probes depicted in Figure 1C or by staining with EtBr.  Nucleic Acids Research, 2009, Vol. 37, No. 4 1129 activity of Rrp44 and whether other nuclease activities are substrate (50 -GGCCCCGGGC CCCGUAGAAA AUCU associated with the core exosome? UAGUAA UCCUUCUUAC AUUGCCCGGG GC-30 ) in 10 mM Tris–HCl pH 7.6, 75 mM NaCl, 2 mM DTT, 100 mg/ml BSA, 0.8 U/ml RNasin, 4.5% glycerol, 0.05% MATERIALS AND METHODS Nonidet P40, 0.5 mM MgCl2 and 0, 0.5 or 5 mM MnCl2. In vivo analyses Prior to addition of the labeled RNA, 10 ml reactions were pre-incubated for 10 min at 308C. After an addi- Growth and handling of Saccharomyces cerevisiae were by tional 1–2 h at 308C, reactions were mixed with one standard techniques. Strains were grown at 258C or 308C volume of RNA formamide buffer, heated for 10 min at in YPD or synthetic dropout medium containing 0.67% 658C and separated on a denaturing 12% polyacryla- nitrogen base (Difco) and either 2% glucose or 2% galac- mide/8 M urea sequencing gel. Reaction products were tose. Yeast RNA extraction and northern hybridization visualized by autoradiography. were performed as described (13). Northern signals were generally visualized by autoradiography, with the excep- Affinity purification of yeast exosomes tion of the lighter exposure in Figure 6B, which was generated by a Fuji FLA-5100 PhosphorImager. Oligonu- One step purifications of exosomes on IgG sepharose col- cleotide probes are listed in Table S1. umns were performed as described (18). To isolate exo- Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 some complexes, Csl4-TAP strains were grown in YPD Rrp44 expression plasmids medium to OD600 0.7 at 258C. Each preparation used 1 l of culture, producing 150 ml of the final TEV fraction. The construction of yeast expression plasmids for Rrp44 is Exosomes were purified in buffer TMN150 (20 mM described in detail in (6). Briefly, the RRP44 ORF is fused Tris–HCl pH 7.6, 150 mM NaCl, 0.1% NP-40, 5 mM to a C-terminal tag containing a streptavidin-binding pep- MgCl2) or treated with 800 mM MgCl2 before TEV tide (Strep-tag II), TEV cleavage site and two copies of elution to dissociate endogenous Rrp44. Enzyme concen- the z-domain of protein A (szz-tag) and cloned into the trations were normalized by immunoblotting using an XhoI restriction sites of either pRS316 (URA3) or pRS315 anti-Rrp6 antibody (19) or anti-peptide antibodies raised (LEU2) (14). For both yeast and E. coli expression plas- against Rrp44 and Rrp43 (this study). mids (see below), point mutations were created using the QuikChange kit (Stratagene) and deletion of the PIN- GST pull down assays domain was achieved by PCR using the oligos listed in Table S1. Equal amounts of GST-bait proteins (
1 pmol) were immobilized on glutathione sepharose beads and incu- Deletion/modification of RRP44 and plasmid shuffling bated with purified yeast exosomes (15 ml TEV eluate) for 1 h at 48C in Buffer NB (20 mM Tris–HCl pH 7.6, The BY4741 wild-type (WT) strain was first supplemented 150 mM NaCl, 8.7% glycerol, 0.1% Nonidet P40 and with a plasmid carrying RRP44-szz and a URA3 selective 5 mM MgCl2). The beads were then washed five times marker. The genomic RRP44 ORF was then precisely with buffer NB. The retained proteins were separated on deleted by homologous recombination with a PCR prod- an 8% polyacrylamide/SDS gel and the presence of the uct generated from the pFA6A-kanMX6 deletion cassette (15). Following construction of rrp44D, a second plasmid core exosome was analyzed by immunoblotting with an (pRS315) (14) was introduced that carried LEU2 and antibody specific for Rrp43 (Figure 7D) and Rrp4 (data expressed either WT or mutant Rrp44. The URA3 not shown). Inputs represent 20% of the starting material. plasmid was then counter-selected on plates containing Glycerol gradient analyses 5-fluoroorotic acid (5-FOA), which is converted to the toxic uracil analogue 5-fluoro-uracil by the action of Yeast lysate from 27.5 ODs of cells was prepared in buffer Ura3. RRP44 was modified in W303 to provide control TMN150 containing 1 mM dithioerythritol and complete by the GAL promoter by integration of a PCR product EDTA-free protease inhibitors (Roche) and loaded on a generated from pYM-N27 (16). Oligonucleotides used in linear 4-ml 10 to 30% (w/v) glycerol gradient. After cen- generation of PCR cassettes are described in Table S1. trifugation for 17 h at 45 000 rpm in a Beckman SW60 rotor, the gradient was harvested manually from the top. Overexpression and purification of recombinant proteins Proteins were isolated from the gradient fractions by ace- E. coli strain BL21(DE3)pLysS (Stratagene) was trans- tone precipitation, separated on an 8% polyacrylamide/ formed with plasmids encoding WT or mutant GST- SDS gel and analyzed by immunoblotting. Rrp44 [derived from GST-Dis3sc (17)] or GST alone (pGEX-4T1). Expression, cell lysis and single-step purifi- RESULTS cation on glutathione sepharose was performed as pre- viously described (6). Yeast strains lacking the exonuclease activity of Rrp44 are viable and retain partial exosome function In vitro assays To assess the effects of the loss of the enzymatic activity Ribonuclease assays were performed with 200 fmol recom- of Rrp44 in vivo, we modified strain BY4741 to binant GST-Rrp44 and 10 fmol 50 - or 30 -end-labeled A30 express HA-tagged Rrp44 under control of the repressible RNA or 150 fmol 50 -end-labeled 52-nt stem loop RNA GAL10 promoter (Table S1), allowing depletion of 1130 Nucleic Acids Research, 2009, Vol. 37, No. 4 endogenous Rrp44. This strain was transformed with plas- The exonuclease activity of Rrp44 appears to play mids expressing either WT-Rrp44 or Rrp44 lacking exo- an important role in maturation of the 7S pre-rRNA, nuclease activity (Rrp44-exo, Figure 1A) under the control and in degradation of the excised 50 -ETS pre-rRNA of the endogenous RRP44 promoter, or with the empty region and truncated 5S rRNA. In contrast, the exonu- cloning vector (pRS316). To allow purification of exosome clease activity is less required for the, presumably indirect, complexes to test for in vitro exonuclease activity, the role of Rrp44 in early pre-rRNA processing steps. plasmid-expressed proteins also carried C-terminal fusions However, strains lacking the exonuclease activity of with a tag containing the streptavidin-binding peptide, Rrp44 are viable and showed less RNA processing TEV cleavage site and two copies of the z-domain of pro- defects than the Rrp44-depleted strain on the pre-rRNA tein A (szz-tag; see Materials and methods section and substrates. Table S1). The growth of individual transformants was then ana- Loss of the exonuclease activity of Rrp44 has additive lyzed under repressive conditions on SD -His/-Ura effects with loss of Rrp6 medium at 308C (Figure 1B). Growth of the strain carry- Constructs expressed under the GAL promoter are ing the empty vector was progressively reduced after never fully repressed, and so a low level of Rrp44 will transfer to glucose. In contrast, growth was fully restored always be expressed in a GAL::rrp44 strain. To avoid Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 by expression of WT Rrp44. A reduced rate of growth was this problem, the RRP44 ORF was precisely deleted in maintained by expression of the catalytically inactive strain BY4741 supplemented with a plasmid carrying Rrp44-exo (Figure 1B). After prolonged growth in glucose RRP44 and a URA3 selective marker. Following construc- medium (24 h), the doubling time of strains expressing tion of rrp44D, a second plasmid was introduced that only Rrp44-exo was
3 h when compared to the WT dou- carried LEU2 and expressed either Rrp44 or Rrp44-exo. bling time of
2 h (data not shown). This indicates that Mitotic segregants that had lost the URA3 plasmid the essential function of Rrp44 in vivo does not require its containing wild type RRP44 were then isolated on exonuclease activity. plates containing 5-FOA, which selectively kills cells Genetic depletion of Rrp44 or any other core exosome expressing Ura3. The rrp44D strains showed an increased component results in characteristic defects in 30 -matura- doubling time when complemented by expression of tion of the 5.8S rRNA from the 7S pre-rRNA and in Rrp44-exo (3 h when compared to 2 h for WT-Rrp44, degradation of the excised 50 -external transcribed spacer Figure 2), similar to the results obtained with region (50 -ETS) of the pre-rRNA (20–23). Exosome GAL::rrp44 strains (Figure 1B). mutants were also reported to accumulate a 30 -truncated Since Rrp44 is essential for viability, whereas its exonu- and polyadenylated fragment of the 5S rRNA (24). clease activity is largely dispensable, it seemed likely that it Northern analysis of the GAL::rrp44 strain also expres- was partially redundant with the activities of one or more sing WT Rrp44 (Figure 1D, lanes 1 and 2) revealed similar other nucleases. An obvious possibility was the nuclear, levels of 30 extended pre-5.8S rRNA and the 50 -ETS in exosome-associated exonuclease Rrp6. Indeed, the galactose (GAL, lane 1) and glucose (GLU, lane 2) rrp44-exo mutation was reported to result in synthetic media. Expression of only Rrp44-exo resulted in the accu- lethality with an rrp6D (4). However, this was not mulation of truncated 5S rRNA (labeled as 5S) and the 7S pre-rRNA plus truncated fragments (Figure 1D, lane 4). Accumulation of longer 30 -extended 5.8S species was greater in the Rrp44-depleted strain, whereas the shortened 6S pre-rRNA was reduced, indicating a greater inhibition of processing following depletion of Rrp44 than in the rrp44-exo strain. Accumulation of the full length 50 -ETS was greater in the Rrp44-depleted strain (Figure 1D, lane 6) than in the rrp44-exo strain (Figure 1D, lane 4), which also showed prominent 50 -ETS fragments that were absent from the Rrp44-depleted strain. These observations are consistent with residual degradation activity associated with Rrp44-exo. Rrp44-depleted strains also exhibit defects at earlier steps in pre-rRNA maturation on the pathway of 18S rRNA synthesis (25). These effects are likely to be indirect, since 18S rRNA synthesis involves only endonuclease activities, and similar defects are seen in many strains with late-acting defects in the 25S and 5.8S rRNA synthe- sis pathway (26). Northern analyses of high molecular Figure 2. Growth analysis of yeast strains expressing Rrp44-exo. Yeast weight pre-rRNA precursors (35S, 27SA2 pre-rRNA and strains carrying either rrp44D or rrp44D rrp6D were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N muta- the aberrant 23S RNA) showed only very modest defects tion (exo). To analyze growth, strains were grown in selective liquid in the strain expressing Rrp44-exo (Figure S1, and see glucose medium at 308C (rrp44D) or 258C (rrp44D rrp6D), which are below). their respective optima.  Nucleic Acids Research, 2009, Vol. 37, No. 4 1131 observed in our strains (Figures 2 and 3). To test for was then analyzed on SD -His/-Leu medium at 258C synthetic lethality between rrp44-exo and rrp6D, the (Figure 2). The loss of Rrp44-exonuclease activity RRP6 ORF was first deleted in the rrp44D strain comple- reduced the growth of RRP6 and rrp6D by a similar pro- mented with a plasmid carrying RRP44 and a URA3 portion, relative to the same strains expressing Rrp44 selective marker (see above). A second plasmid (pRS315) (Figure 2). carrying LEU2 and expressing either Rrp44 or Rrp44-exo Northern analysis was further used to compare the was subsequently introduced and the URA3 plasmid RNA processing phenotypes of rrp44D rrp6D double was then counter-selected on 5-FOA containing SD mutant strains complemented by plasmids expressing -His/-Leu plates. The growth of individual transformants either WT Rrp44 or Rrp44-exo (Figure 3). Yeast strains Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 Figure 3. The rrp44-exo mutation is not synthetically lethal with deletion of Rrp6. (A) and (B). In vivo analyses of yeast strains expressing Rrp44-exo in the presence or absence of the exosome components Rrp6 or Rrp41. RNA was separated on an 8% polyacrylamide/8 M urea gel (A) or an 1% agarose/glyoxal gel (B) and analyzed as described in Figure 1D. 1132 Nucleic Acids Research, 2009, Vol. 37, No. 4 carrying rrp6D exhibit a distinctive accumulation of the 30 -extended precursor 5.8S + 30 (27) (Figure 3A, lane 3), which is found as a short (S) and a 50 -extended long (L) form. Expression of only Rrp44-exo in the rrp6D strain leads to accumulation of a longer form of this precursor (Figure 3A, lane 4). To confirm the identity of the extended 5.8S species, primer extension was performed using a probe located across the 5.8S-ITS2 boundary (data not shown). This revealed identical 50 ends, showing that the species detected in the rrp6D rrp44-exo double mutant is 30 extended by
10 nt relative to the 5.8S + 30 RNA seen in rrp6D single mutant strains. Loss of Rrp6 has little effect on degradation of the excised 50 -ETS (Figure 3A, lane 3) and the phenotype of the rrp6D rrp44-exo strain (Figure 3A, lane 4) resembled that of rrp44-exo alone (Figure 3A, lane 2). Both 30 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 extended and truncated forms of the box C + D snoRNA snR13 were previously observed in strains with defects in nuclear RNA surveillance (28,29). These forms of snR13 were also detected in both the rrp6D and rrp44-exo single mutants (Figure 3A, lanes 3 and 2), with stronger accumulation in the double mutant strain (Figure 3A, lane 4). High molecular weight RNA was also analyzed (Figure 3B). The rrp6D mutation alone resulted in increased levels of the 27SA2 pre-rRNA, the first commit- ted intermediate on the pathway of 5.8S and 25S rRNA synthesis. The aberrant 23S, 21S and 17S RNAs were also accumulated (Figure 3B, lane 3); these are known targets for degradation by Rrp6 and the TRAMP5 polyadenyla- tion complex (24,25,30). In contrast, rrp44-exo alone reduced the level of 27SA2 but had little effect on 23S (Figure 3B, lane 2). The double mutant showed an inter- mediate phenotype. Since the exonuclease activity of Rrp44 is dispensable for growth, even in the absence of Rrp6, we considered the possibility that the essential role of the other, appar- ently non-catalytic components of the core exosome might lie in restraining and controlling an otherwise over- promiscuous exonuclease activity of Rrp44. To test this model, the core exosome component Rrp41 was placed under the control of a GAL promoter in the rrp44D strain expressing either intact Rrp44 or Rrp44-exo. However, following transfer to glucose medium, the GAL::rrp41 strains showed similar growth inhibition upon expression of Rrp44 or Rrp44-exo (data not Figure 4. The Rrp44-exo mutation, but not the Rrp44-endo mutation, is shown). Northern analyses (Figure 3A and B, lanes 5–8) synthetically lethal with loss of Xrn1. (A) In vivo analysis of Gal::rrp44 also indicated that the loss of Rrp44 exonuclease activity strains expressing WT-Rrp44 or Rrp44-exo in the absence of the cyto- did not suppress or give synergistic defects when combined plasmic 50 –30 exonuclease Xrn1. Strains were streaked for single colo- with depletion of Rrp41. We conclude that the pheno- nies on selective plates containing either 2% galactose or 2% glucose type of Rrp41 depletion is neither dependent on, nor and incubated at 308C. (B) In vivo analysis of rrp44D strains expressing WT or mutant Rrp44 in the absence of Xrn1. Strains were streaked for attenuated by, the exonuclease activity of Rrp44. single colonies on selective plates with or without 5-FOA, which selects for loss of the plasmid carrying URA3 and WT-RRP44, and incubated Rrp44 exonuclease activity is essential in the absence of the at 258C. cytoplasmic 5’–3’ exonuclease Xrn1 Since the exonuclease activity of Rrp44 was apparently not strongly redundant with Rrp6, we tested for synthetic synthetically lethal with the cytoplasmic cofactors for lethal interactions with other exonucleases. the exosome, due to synergistic inhibition of mRNA deg- The non-essential, 50 –30 -exonuclease Xrn1 (31) plays radation (32,33). Deletion of XRN1 was synthetically a major role in cytoplasmic mRNA turnover and is lethal with rrp44-exo (Figure 4A). We conclude that loss  Nucleic Acids Research, 2009, Vol. 37, No. 4 1133 of the 30 -exonuclease activity of Rrp44 is synthetic-lethal presence of Mn2+, both WT and mutant proteins cleaved with loss of the cytoplasmic 50 –30 exonuclease Xrn1, prob- the RNA, generating very similar degradation products ably due to synergistic inhibition of mRNA degradation. (lanes 5 and 6). From these results, we concluded that In contrast, loss of the PIN-domain associated endonu- Rrp44 harbors two distinct ribonuclease activities, which clease activity of Rrp44 (see below) was not synthetic- respond differently to Mn2+ ions under in vitro condi- lethal with xrn1D (Figure 4B). tions. The presence of Mn2+ inhibited 30 -exonuclease In addition to Rrp44, yeast contains two other proteins activity, whereas a second activity was stimulated. with homology to the RNase II family. Of these, Dss1 is To further characterize the novel activity, we performed mitochondrial and seemed unlikely to function redun- in vitro nuclease assays using the exonucleolytically inac- dantly with Rrp44. In contrast, Ssd1 is localized to the tive Rrp44-exo mutant and 50 - and 30 -labeled A30 RNA cytoplasm (34,35) and shows genetic interactions consis- substrates (Figure 5B). Rrp44-exo generated a ladder of tent with functions in RNA turnover and/or surveillance degradation intermediates from both substrates in the (36,37). Laboratory strains of yeast are polymorphic for absence of added Mn2+ ions (lanes 2 and 6), likely due Ssd1 synthesis (38); strains derived from S288c that were to co-purification of a low level of Mn2+ bound to the used for the systematic sequencing project and construc- recombinant protein, but the degradation activity was tion of the gene deletion collection, including BY4741 that strongly stimulated in the presence of added Mn2+ Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 we used for our initial functional analyses of Rrp44, (lanes 4 and 8). The ability of the Rrp44-exo mutant to harbor the full-length protein (Ssd1-v), whereas the generate degradation intermediates from both 30 -labeled widely used W303 strain expresses a truncated version of and 50 -labeled substrates shows that it either carries both the protein (Ssd1-d). In comparison to GAL::rrp44 strains 50 -exonuclease and 30 -exonuclease activities or, more derived from BY4741, expression of Rrp44-exo in the likely, possesses an endonuclease activity. W303 background consistently supported slightly less effi- To assess if this potential endonuclease activity was cient growth when intact Rrp44 was depleted by incuba- associated with the PIN domain of Rrp44, point muta- tion on glucose medium (data not shown). To determine tions were introduced at each of the four conserved whether this was due to the lack of intact Ssd1, the full- active-site amino acids (D91N, E120Q, D171N, D198N), to length form of Ssd1 was expressed from a plasmid. create the Rrp44-endo mutant. In addition, the Rrp44-exo However, this failed to clearly improve growth of W303 mutation was combined with the four PIN-domain muta- strains expressing only Rrp44-exo. tions (Rrp44-endo–exo) and with a point mutation in the Strain differences between BY4741 and W303 have a S1 RNA-binding domain (Rrp44-exo-S1) (6). modest but reproducible impact on sensitivity to loss of In vitro nuclease assays were performed using recombi- Rrp44 exonuclease activity. Similar genetic background nant, GST-tagged proteins and a 50 -labeled substrate effects may be responsible for the fact that we found RNA derived from the 30 region of the mouse 5.8S strains lacking the exonuclease activity of both Rrp44 rRNA, which has a well defined, stable terminal stem and Rrp6 to be viable in BY4741, whereas they were pre- structure (Figure 5C and D). WT Rrp44 (Figure 5C, viously reported to be synthetic lethal in strain BMA 64, lane 2) partially degraded the substrate to a 10 nt product. which is related to W303 (4). In contrast, Rrp44-exo generated a set of fragments with end-points in the loop-region of the substrate RNA The PIN domain of Rrp44 shows endonuclease activity (Figure 5C, lane 3). These fragments were not generated by either the Rrp44-endo (Figure 5C, lane 4) Comparison of the RNA processing phenotype of or Rrp44-endo–exo proteins (lane 5), indicating that Rrp44-depletion to that shown by strains expressing their formation required a functional PIN domain. Rrp44-exo (Figure 1D) suggested that some residual Rrp44-exo-S1 did generate the loop fragments, but nuclease activity remained. Recent analyses of the PIN lacked the major 10 nt product, suggesting that S1 domain proteins, human Smg6 and yeast Swt1, had RNA-binding activity might be more important for the revealed that robust in vitro endonuclease activity required exonuclease activity of Rrp44 than for endonuclease activ- the presence of manganese ions (8,9). Four conserved ity. A prominent product that is truncated by
10 nt was acidic residues (Aspartate/D or Glutamate/E) coordi- detected in some samples. This appears to be due to a nate the metal ion in the active site. All four residues are contaminating nuclease that is associated with purification present in the N-terminal PIN domain of Rrp44 of GST-fusion constructs from E. coli, as the same band (Figure 1A and see below) and we therefore assessed was seen in analyses of other fusion proteins (data not whether Rrp44 also exhibited endonuclease activity in shown). The truncated fragment appears to be very sus- the presence of Mn2+ (Figure 5). ceptible to degradation by the exonuclease activity of In vitro nuclease assays were first performed in the Rrp44, giving rise to the observed variations in signal. absence or presence of 5 mM Mn2+ using recombinant, The PIN domain alone did not show in vitro activity. GST-tagged proteins and a 50 -end-labeled A30 RNA sub- However, when purified from E. coli the PIN domain strate (Figure 5A). Protein preparations used are shown in co-purified, apparently stoichiometrically, with the bacte- Figure S2. Consistent with previous results (6), WT Rrp44 rial chaperone GroEL (indicated with an asterisk in (lane 2) exhibited strong 30 -exonucleolytic activity in the Figure 7B), strongly indicating that it is largely misfolded. absence of Mn2+, completely degrading the substrate to a We conclude that recombinant Rrp44 shows an endonu- 4 nt product. In contrast, the Rrp44-exo mutant showed clease activity that requires Mn2+ ions and the metal- little degradation activity (lane 3). However, in the binding amino acids of the PIN domain. 1134 Nucleic Acids Research, 2009, Vol. 37, No. 4 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 Figure 5. Recombinant Rrp44 exhibits two distinct ribonuclease activities in vitro. (A) The ribonuclease activity of recombinant GST-Rrp44 is altered by the presence of manganese ions. Two hundred fmol recombinant protein (WT or Rrp44-exo) and 10 fmol 50 -end-labeled A30 RNA substrate were incubated for 60 min at 308C in the presence of 0.5 mM MgCl2 and in the absence or presence of 5 mM MnCl2. Reaction products were separated on a 12% polyacrylamide/8 M urea gel and visualized by autoradiography. Control: mock-digested substrate lacking recombinant protein. (B) Ribonuclease activity of recombinant GST-Rrp44-exo on 50 -end-labeled (lanes 1–4) or 30 -end-labeled (lanes 5–8) A30 RNA substrate. Assay condi- tions were as described in (A). (C) Endonucleolytic activity of recombinant GST-Rrp44 on a 50 -end-labeled RNA substrate with a stable terminal stem structure. Two hundred femtomoles recombinant protein (WT or mutants, see below) and 150 fmol RNA were incubated for 2 h at 308C in the presence of 0.5 mM MgCl2 and 0.5 mM MnCl2. Reaction products were analyzed as described above. The regions corresponding to the stem structure are indicated with black lines on the gel (C) and cartoon (D). The region corresponding to the loop is indicated in gray. The asterisks mark two degradation products of unclear origin that occur when the substrate is incubated with any form of recombinant GST-Rrp44 or other GST-tagged proteins (data not shown). Rrp44-exo: Rrp44 with D551N mutation. Rrp44-endo: Rrp44 with four individual amino-acid exchanges (D91N, E120Q, D171N and D198N) in the metal-coordinating centre of the N-terminal PIN domain. Rrp44-endo–exo: Rrp44 with 4 PIN mutations plus D551N. Rrp44-exo-S1: Rrp44 with D551N plus a G916E mutation in the S1 RNA-binding domain.  Nucleic Acids Research, 2009, Vol. 37, No. 4 1135 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 Figure 6. The N-terminal PIN domain in Rrp44 harbors endonuclease activity in vivo. (A) Northern analysis of pre-rRNA processing in the GAL::rrp44 strain transformed with plasmids expressing either WT or mutant Rrp44 protein, or an empty vector pRS315. The mutants analyzed are rrp44-exo, rrp44-endo and rrp44-endo–exo (see Figure 5). RNA was isolated from GAL::rrp44 strains grown at 308C under permissive conditions (GAL) and 10 h after transcriptional repression (GLU). RNA was separated on an 8% polyacrylamide/8 M urea gel and analyzed as described in Figure 1D. (B) Northern analysis of pre-rRNA processing in rrp44D (lanes 1–3) or rrp44Drrp6D (lanes 4–6) strains transformed with a plasmid expressing either WT or mutant Rrp44 protein. Strains were grown at 308C (rrp44D) or 258C (rrp44D rrp6D) and RNA was analyzed as in (A). Two different exposures are shown for the probe recognizing precursors of the 5.8S rRNA (#020). The effects of the rrp44-endo mutation were also In contrast, expression of Rrp44-endo–exo failed to sup- tested on viability. In the GAL::rrp44 strain, expression port growth (data not shown), indicating that the of Rrp44-endo clearly supported growth when WT endonuclease and exonuclease activities of Rrp44 Rrp44 was depleted by growth on glucose medium. share some redundant essential function. In plasmid 1136 Nucleic Acids Research, 2009, Vol. 37, No. 4 shuffle experiments, expression of Rrp44-endo, but not Rrp44, but predominantly sedimented as free protein. In Rrp44-endo–exo, supported viability in an rrp44D rrp6D Rrp44-depleted cells, Rrp43 becomes enriched in double mutant strain (data not shown, but see Figure 6B,
60S gradient fractions, reflecting stabilized exosome lane 6). A low number of rrp44D strains complemented association with pre-ribosomes (L. Milligan and D.T., by the plasmid expressing Rrp44-endo–exo could be iso- unpublished results), giving rise to the strong signal in lated by plasmid shuffle (data not shown). Rare cells are the pellet fractions on the gradient shown. therefore able to adapt to loss of both the endonuclease Binding of Rrp44 to the core exosome was also assessed and exonuclease activities of Rrp44 by epigenetic or phys- by in vitro binding assays using recombinant GST- iological mechanisms. tagged Rrp44 constructs expressed in E. coli (Figure 7B) Northern analyses were performed on the GAL::rrp44 and core exosome purified from yeast, with or without strain expressing Rrp44-endo and Rrp44-endo–exo during removal of Rrp44 by washing with 800 mM MgCl2 Rrp44 depletion (Figure 6A). These showed that the (Figure 7C). The GST-tagged constructs were bound to 50 -extended forms of the 5.8S rRNA and truncated glutathione sepharose and incubated with the exosome forms of the 50 -ETS observed in strains expressing preparations. The association of the exosome with the Rrp44-exo were suppressed in the rrp44-endo strain GST-fusion proteins was assessed by western blotting and altered in the rrp44-endo–exo strain. In particular, using antibodies specific for Rrp43 (Figure 7D) and Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 50 -ETS fragments accumulating in the rrp44-exo strain Rrp4 (data not shown). Exosome binding to the full- (Figure 6A, lane 4) were clearly reduced in strains expres- length Rrp44 (FL) (Figure 7D, lane 2) was substantially sing Rrp44-endo–exo (Figure 6A, lane 8). This suggested stronger than to Rrp44 lacking the N-terminal PIN that the endonuclease activity of Rrp44 is involved in for- domain (
PIN) (Figure 7D, lane 4), which was not mation of intermediate species that are stabilized by loss above the GST background (Figure 7D, lane 6). of Rrp44 30 -exonuclease activity. The longer form of the Notably, exosome binding was not affected by four indi- 5.8S + 30 RNA seen in the rrp6D strain expressing vidual mutations in the PIN domain (Rrp44-endo, see Rrp44-exo (Figure 6B, lane 5) was not observed in the Figure 5) (Figure 7D, lane 3). The PIN domain alone rrp6D strain expressing Rrp44-endo (Figure 6B, lane 6). (PIN) (Figure 7D, lane 5) gave a lower signal, but this Accumulation of the truncated form of the 5S rRNA, seen clearly was above the GST background. As noted above, in rrp44-exo strains, was not suppressed in the rrp44-endo– the low signal may reflect poor folding of the isolated PIN exo strain. However, the truncated 5S was accumulated in domain, as suggested by its copurification with bacterial rrp6D strains, even in the presence of the rrp44-endo GroEL. The signal from the Rrp44-depleted exosome mutation. (exosome –44) was stronger than for exosome purified at Expression of only Rrp44 lacking both the endonu- lower salt concentrations (exosome +44). However, some clease and exonuclease activity resulted in a phenotype binding to Rrp44 was seen even without the high-salt that closely resembled that seen on depletion of Rrp44 wash, suggesting that Rrp44 is substoichiometric in the (vector control in Figure 6A, lane 10). Together the data purified exosome. indicate that the PIN domain of Rrp44 exhibits endonu- We conclude that the PIN domain of Rrp44 plays a clease activity in vitro and in vivo. dual role in that it harbors endonuclease activity and also functions in tethering Rrp44 to the remaining nine The PIN domain links Rrp44 to the core exosome subunit core of the exosome. The archaeal exosome lacks a homologue of Rrp44, and yeast Rrp44 can be removed from the remaining nine DISCUSSION components of the exosome by washing with high salt. This indicates that Rrp44 associates with a highly stable, Genetic depletion of RRP44 is lethal (21), whereas strains nine component exosome core structure. A two-hybrid expressing the Rrp44-exo mutant are viable with only a interaction was detected between the PIN domain of modest growth defect, at least in some strain backgrounds, human Rrp44 and the RNase PH-homologue OIP2 (the showing that its exonuclease activity is dispensable for human homologue of yeast Rrp43) (39) suggesting that growth. It is, of course, difficult to be certain that any the PIN domain might be involved in protein–protein mutation fully inhibits enzymatic activity in vivo. interactions that tether Rrp44 to the core structure. This However, this seems probable, as the D551N mutation model would be consistent with the absence of PIN (rrp44-exo) abolished detectable in vitro activity on more domains from E. coli RNase R and II (Figure 1A), than five different substrates (4–6) while the equivalent which function as monomers. mutation (D209N) abolished the activity of E. coli RNase The association of Rrp44 with the exosome core was II (40). initially tested by glycerol gradient centrifugation of The exonuclease activity of Rrp44 does play an impor- yeast cell lysates (Figure 7A). WT, protein A-tagged tant role in pre-rRNA processing, participating in both Rrp44 largely co-sedimented with the core exosome com- the maturation of the 7S pre-rRNA to 5.8S rRNA and ponent Rrp43 in
14S complexes (fractions 14–17), with a the degradation of the excised 50 -ETS region. However, on small peak corresponding to the position of free both substrates the phenotype of strains expressing Rrp44 (fractions 7–8). In contrast, protein A-tagged only Rrp44-exo was substantially weaker than that seen Rrp44 lacking the PIN-domain (
aa 86–203), was in strains lacking Rrp44. The only substrate tested expressed at levels substantially below that of WT that showed apparently identical accumulation in  Nucleic Acids Research, 2009, Vol. 37, No. 4 1137 Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 Figure 7. The N-terminal PIN domain in Rrp44 mediates protein–protein interactions. (A) Sedimentation behavior of WT and Rrp44-
PIN on glycerol gradients. Gal::rrp44 strains transformed with plasmids encoding protA-tagged Rrp44 [full-length (WT) or
PIN] were grown on glucose for 8.5 h to deplete endogenous Rrp44. Protein extracts from 27.5 ODs of cells were then separated on linear 10–30% glycerol gradients and analyzed by western blotting. (B) Recombinant, GST-tagged WT Rrp44 (WT), Rrp44-endo (endo), Rrp44-
PIN (
PIN), the PIN domain alone (PIN) and free GST were expressed in E. coli and purified on a glutathione sepharose column. The proteins (indicated by arrowheads) were separated on an 8% polyacrylamide/SDS gel and stained with Coomassie blue. Asterisk: E. coli GroEL. (C) Western blot analysis of TAP-purified exosomes from Csl4-TAP strains grown at 258C. Exosomes were purified on IgG sepharose at 150 mM NaCl (lane 1) or treated with 800 mM MgCl2 to dissociate endogenous Rrp44 (lane 2). TEV eluates (2.5 ml) were separated on an 8% polyacrylamide/SDS gel and analyzed by immunoblotting with anti-Rrp44, anti-Rrp6 and anti-Rrp43 antibodies. (D) GST-pull down experiment. The GST- tagged Rrp44 constructs or free GST (B) were coupled to glutathione sepharose beads and incubated with purified yeast exosomes (C). Bound material was eluted under denaturing conditions, separated on an 8% polyacrylamide/SDS gel and analyzed by immunoblotting with an anti-Rrp43 antibody. Rrp44-depleted and rrp44-exo strains was a 30 -truncated strain background, even though both are widely used form of the 5S rRNA, which was previously identified as a ‘wild-type’ strains. We tested W303 because it expresses TRAMP/exosome substrate (24). only a truncated form of the RNase II-related protein Strain heterogeneity was observed in sensitivity to Ssd1, and we speculated that this might show genetic the rrp44-exo mutation, which conferred a stronger interactions with loss of the exonuclease activity of growth phenotype in strain W303 than in a BY4741 Rrp44. In the event, expression of full-length Ssd1 failed 1138 Nucleic Acids Research, 2009, Vol. 37, No. 4 to suppress the sensitivity of W303 to the loss of Rrp44 participates in the degradation of the excised spacer exonuclease activity. region following A0 cleavage. Several early acting ribo- The essential function of Rrp44 might be the mainte- some synthesis factors are released from the pre-ribosomes nance of the structure of the exosome complex or bind- in association with the excised 50 -ETS (44). Very efficient ing to either substrates or cofactors. Recombinant degradation of the 50 -ETS is therefore likely to be impor- GST-Rrp44-exo protein was able to bind to RNA (6), tant for the release and recycling of ribosome synthesis and the yeast exosome containing Rrp44-exo might thus factors. still be able to recruit RNAs that are normally substrates Analyses in vivo and in vitro showed that the PIN for Rrp44. Substrates recognized and bound by domain of Rrp44 was both necessary and sufficient for Rrp44-exo might then be degraded by other nucleases. association with the 9 subunit exosome core structure. Loss of Rrp44 exonucleolytic activity is synthetic lethal Previous EM analyses indicated that both the Rrp44 N- (sl) with deletion of the nonessential cytoplasmic 50 –30 exo- terminus (where the PIN domain is located) and the nuclease Xrn1, due to the synergistic inhibition of cyto- C-terminal region form interactions with the exosome plasmic mRNA degradation (33). This indicates that the core (45). The data presented here indicate that the rrp44-exo mutation blocked the 30 degradation of at least N-terminal interactions are more crucial for stable bind- some mRNA species in vivo. In contrast, BY4741 strains ing. Alterations in the relative positions of the N- and Downloaded from http://nar.oxfordjournals.org/ at University of Newcastle on April 28, 2012 expressing only the Rrp44-exo mutant were viable in the C-terminal regions of Rrp44 seen on association with absence of the other exosome-associated 30 -exonuclease the core exosome are proposed to be crucial for regulation Rrp6. This finding differs from previous observations of its exonuclease activity (45), and interactions between made in a W303-related strain (BMA 36) (4), presumably the C-terminal region of Rrp44 and the exosome may play reflecting the differences in strain background. Recent an important role in this regulation. analyses (41) indicate that functional interactions between Within Rrp44, the PIN domain appears to have a dual Rrp6 and Rrp44 are important for only a subset of Rrp6 role, acting to link Rrp44 to the remainder of the exosome substrates. in addition to its apparent cleavage activity. This evolu- The exonuclease activities of the exosome in the nucleus tion of the region therefore appears to play a crucial part (Rrp44 and Rrp6) likely form part of a redundant network in the functional differences between bacterial RNase R of 30 -exonucleases, including the proteins of the Rex and eukaryotic Rrp44. The structure of the exosome core family (42), which could, for instance, be needed to fine- is related to that of bacterial PNPase (5,46,47). In E. coli, tune the balance between pre-ribosomal RNA processing PNPase can associate with RNase E and other proteins in and decay. In the cytoplasm, such a fine balance might the degradosome complex, that shows both 30 -exonuclease not be necessary, since RNA decay is the predominant and endonuclease activity (48). The finding that the pathway. eukaryotic exosome also possesses both 30 -exonuclease The differences in RNA processing phenotypes between and endonuclease activity underlines the conservation of strains depleted of Rrp44 and those expressing Rrp44-exo, the RNA processing machinery. suggested that Rrp44 might harbor additional activities. Recent reports of manganese-dependent endonuclease activities associated with other PIN-domain proteins SUPPLEMENTARY DATA prompted us to determine whether this might also be the Supplementary Data are available at NAR Online. case for Rrp44. The endonuclease activity of Rrp44 failed to be observed in previous analyses, probably because in the absence of manganese the exonuclease activity is much ACKNOWLEDGEMENT more prominent. The concentrations of 0.5 or 5 mM Mn2+ that were used to inhibit exonuclease activity We thank J. Houseley for yeast strains and reagents and and stimulate endonuclease activity of Rrp44 are much critical reading of the manuscript. greater than the physiological concentration (43). It seems likely that the in vivo endonuclease activity of FUNDING Rrp44 is stimulated by one or more of the many exosome cofactors, allowing it to act at lower Mn2+ concentra- This work was supported by the Wellcome Trust (to D.T.) tions. However, we cannot fully exclude the possibility and BBSRC (to J.D.B.), a long-term HFSP fellowship (to that the PIN domain primarily functions in RNA C.S.) and a PhD studentship from the BBSRC (to E.L.). substrate binding in vivo, with a non-physiological cleav- Funding for open access charge: Wellcome Trust. age activity that is induced by high Mn2+ levels in vitro. The range of in vivo substrates for the activity of the Conflict of interest statement. None declared. Rrp44 PIN domain remains unclear but comparison of the RNA processing phenotypes of strain expressing REFERENCES Rrp44-exo and Rrp44-endo–exo strongly indicates that these are likely to include the 50 -ETS region of the pre- 1. Allmang,C., Petfalski,E., Podtelejnikov,A., Mann,M., Tollervey,D. rRNA. However, the putative target sites do not appear to and Mitchell,P. (1999) The yeast exosome and human PM-Scl are related complexes of 30 –50 exonucleases. 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