Yeast strains lacking the exonuclease activity of Rrp44 are viable and retain partial exosome function
To assess the effects of the loss of the enzymatic activity of Rrp44 in vivo, we modified strain BY4741 to express HA-tagged Rrp44 under control of the repressible GAL10 promoter (Table S1), allowing depletion of endogenous Rrp44. This strain was transformed with plasmids expressing either WT-Rrp44 or Rrp44 lacking exonuclease activity (Rrp44-exo, A) under the control of the endogenous RRP44 promoter, or with the empty cloning vector (pRS316). To allow purification of exosome complexes to test for in vitro exonuclease activity, the plasmid-expressed proteins also carried C-terminal fusions with a tag containing the streptavidin-binding peptide, TEV cleavage site and two copies of the z-domain of protein A (szz-tag; see Materials and methods section and Table S1).
The growth of individual transformants was then analyzed under repressive conditions on SD -His/-Ura medium at 30°C (B). Growth of the strain carrying the empty vector was progressively reduced after transfer to glucose. In contrast, growth was fully restored by expression of WT Rrp44. A reduced rate of growth was maintained by expression of the catalytically inactive Rrp44-exo (B). After prolonged growth in glucose medium (24 h), the doubling time of strains expressing only Rrp44-exo was ~3 h when compared to the WT doubling time of ~2 h (data not shown). This indicates that the essential function of Rrp44 in vivo does not require its exonuclease activity.
Genetic depletion of Rrp44 or any other core exosome component results in characteristic defects in 3′-maturation of the 5.8S rRNA from the 7S pre-rRNA and in degradation of the excised 5′-external transcribed spacer region (5′-ETS) of the pre-rRNA (20–23
). Exosome mutants were also reported to accumulate a 3′-truncated and polyadenylated fragment of the 5S rRNA (24
Northern analysis of the GAL::rrp44 strain also expressing WT Rrp44 (D, lanes 1 and 2) revealed similar levels of 3′ extended pre-5.8S rRNA and the 5′-ETS in galactose (GAL, lane 1) and glucose (GLU, lane 2) media. Expression of only Rrp44-exo resulted in the accumulation of truncated 5S rRNA (labeled as 5S*) and the 7S pre-rRNA plus truncated fragments (D, lane 4). Accumulation of longer 3′-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 5′-ETS was greater in the Rrp44-depleted strain (D, lane 6) than in the rrp44-exo strain (D, lane 4), which also showed prominent 5′-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 synthesis pathway (26
). Northern analyses of high molecular weight pre-rRNA precursors (35S, 27SA2
pre-rRNA and the aberrant 23S RNA) showed only very modest defects in the strain expressing Rrp44-exo (Figure S1, and see below).
The exonuclease activity of Rrp44 appears to play an important role in maturation of the 7S pre-rRNA, and in degradation of the excised 5′-ETS pre-rRNA region and truncated 5S rRNA. In contrast, the exonuclease activity is less required for the, presumably indirect, role of Rrp44 in early pre-rRNA processing steps. However, strains lacking the exonuclease activity of Rrp44 are viable and showed less RNA processing defects than the Rrp44-depleted strain on the pre-rRNA substrates.
Loss of the exonuclease activity of Rrp44 has additive effects with loss of Rrp6
Constructs expressed under the GAL promoter are never fully repressed, and so a low level of Rrp44 will always be expressed in a GAL::rrp44 strain. To avoid this problem, the RRP44 ORF was precisely deleted in strain BY4741 supplemented with a plasmid carrying RRP44 and a URA3 selective marker. Following construction of rrp44Δ, a second plasmid was introduced that carried LEU2 and expressed either Rrp44 or Rrp44-exo. Mitotic segregants that had lost the URA3 plasmid containing wild type RRP44 were then isolated on plates containing 5-FOA, which selectively kills cells expressing Ura3. The rrp44Δ strains showed an increased doubling time when complemented by expression of Rrp44-exo (3 h when compared to 2 h for WT-Rrp44, ), similar to the results obtained with GAL::rrp44 strains (B).
Figure 2. Growth analysis of yeast strains expressing Rrp44-exo. Yeast strains carrying either rrp44Δ or rrp44Δ rrp6Δ were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N mutation (exo). To analyze growth, strains (more ...)
‘Since Rrp44 is essential for viability, whereas its exonuclease activity is largely dispensable, it seemed likely that it was partially redundant with the activities of one or more other nucleases. An obvious possibility was the nuclear, exosome-associated exonuclease Rrp6. Indeed, the rrp44-exo
mutation was reported to result in synthetic lethality with an rrp6Δ
). However, this was not observed in our strains ( and ). To test for synthetic lethality between rrp44-exo
, the RRP6
ORF was first deleted in the rrp44Δ
strain complemented with a plasmid carrying RRP44
and a URA3
selective marker (see above). A second plasmid (pRS315) carrying LEU2
and expressing either Rrp44 or Rrp44-exo was subsequently introduced and the URA3
plasmid was then counter-selected on 5-FOA containing SD -His/-Leu plates. The growth of individual transformants was then analyzed on SD -His/-Leu medium at 25°C (). The loss of Rrp44-exonuclease activity reduced the growth of RRP6
by a similar proportion, relative to the same strains expressing Rrp44 ().
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 (more ...)
Northern analysis was further used to compare the RNA processing phenotypes of rrp44Δ rrp6Δ
double mutant strains complemented by plasmids expressing either WT Rrp44 or Rrp44-exo (). Yeast strains carrying rrp6Δ
exhibit a distinctive accumulation of the 3′-extended precursor 5.8S + 30 (27
) (A, lane 3), which is found as a short (S) and a 5′-extended long (L) form. Expression of only Rrp44-exo in the rrp6Δ
strain leads to accumulation of a longer form of this precursor (A, 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 5′ ends, showing that the species detected in the rrp6Δ rrp44-exo
double mutant is 3′ extended by ~10 nt relative to the 5.8S + 30 RNA seen in rrp6Δ
single mutant strains.
Loss of Rrp6 has little effect on degradation of the excised 5′-ETS (A, lane 3) and the phenotype of the rrp6Δ rrp44-exo
strain (A, lane 4) resembled that of rrp44-exo
alone (A, lane 2). Both 3′ extended and truncated forms of the box C + D snoRNA snR13 were previously observed in strains with defects in nuclear RNA surveillance (28
). These forms of snR13 were also detected in both the rrp6Δ
single mutants (A, lanes 3 and 2), with stronger accumulation in the double mutant strain (A, lane 4).
High molecular weight RNA was also analyzed (B). The rrp6Δ
mutation alone resulted in increased levels of the 27SA2
pre-rRNA, the first committed intermediate on the pathway of 5.8S and 25S rRNA synthesis. The aberrant 23S, 21S and 17S RNAs were also accumulated (B, lane 3); these are known targets for degradation by Rrp6 and the TRAMP5 polyadenylation complex (24
). In contrast, rrp44-exo
alone reduced the level of 27SA2
but had little effect on 23S (B, lane 2). The double mutant showed an intermediate 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, apparently 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 rrp44Δ 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 shown). Northern analyses (A and B, lanes 5–8) also indicated that the loss of Rrp44 exonuclease activity did not suppress or give synergistic defects when combined with depletion of Rrp41. We conclude that the phenotype of Rrp41 depletion is neither dependent on, nor attenuated by, the exonuclease activity of Rrp44.
Rrp44 exonuclease activity is essential in the absence of the cytoplasmic 5′–3′ exonuclease Xrn1
Since the exonuclease activity of Rrp44 was apparently not strongly redundant with Rrp6, we tested for synthetic lethal interactions with other exonucleases.
The non-essential, 5′–3′-exonuclease Xrn1 (31
) plays a major role in cytoplasmic mRNA turnover and is synthetically lethal with the cytoplasmic cofactors for the exosome, due to synergistic inhibition of mRNA degradation (32
). Deletion of XRN1
was synthetically lethal with rrp44-exo
(A). We conclude that loss of the 3′-exonuclease activity of Rrp44 is synthetic-lethal with loss of the cytoplasmic 5′–3′ exonuclease Xrn1, probably due to synergistic inhibition of mRNA degradation. In contrast, loss of the PIN-domain associated endonuclease activity of Rrp44 (see below) was not synthetic-lethal with xrn1Δ
Figure 4. The Rrp44-exo mutation, but not the Rrp44-endo mutation, is synthetically lethal with loss of Xrn1. (A) In vivo analysis of Gal::rrp44 strains expressing WT-Rrp44 or Rrp44-exo in the absence of the cytoplasmic 5′–3′ exonuclease (more ...)
In addition to Rrp44, yeast contains two other proteins with homology to the RNase II family. Of these, Dss1 is mitochondrial and seemed unlikely to function redundantly with Rrp44. In contrast, Ssd1 is localized to the cytoplasm (34
) and shows genetic interactions consistent with functions in RNA turnover and/or surveillance (36
). Laboratory strains of yeast are polymorphic for Ssd1 synthesis (38
); strains derived from S288c that were used for the systematic sequencing project and construction of the gene deletion collection, including BY4741 that we used for our initial functional analyses of Rrp44, harbor the full-length protein (Ssd1-v), whereas the widely used W303 strain expresses a truncated version of the protein (Ssd1-d). In comparison to GAL::rrp44
strains derived from BY4741, expression of Rrp44-exo in the W303 background consistently supported slightly less efficient growth when intact Rrp44 was depleted by incubation on glucose medium (data not shown). To determine whether this was due to the lack of intact Ssd1, the full-length form of Ssd1 was expressed from a plasmid. However, this failed to clearly improve growth of W303 strains expressing only Rrp44-exo.
Strain differences between BY4741 and W303 have a modest but reproducible impact on sensitivity to loss of Rrp44 exonuclease activity. Similar genetic background effects may be responsible for the fact that we found strains lacking the exonuclease activity of both Rrp44 and Rrp6 to be viable in BY4741, whereas they were previously reported to be synthetic lethal in strain BMA 64, which is related to W303 (4
The PIN domain of Rrp44 shows endonuclease activity
Comparison of the RNA processing phenotype of Rrp44-depletion to that shown by strains expressing Rrp44-exo (D) suggested that some residual nuclease activity remained. Recent analyses of the PIN domain proteins, human Smg6 and yeast Swt1, had revealed that robust in vitro
endonuclease activity required the presence of manganese ions (8
). Four conserved acidic residues (Aspartate/D or Glutamate/E) coordinate the metal ion in the active site. All four residues are present in the N-terminal PIN domain of Rrp44 (A and see below) and we therefore assessed whether Rrp44 also exhibited endonuclease activity in the presence of Mn2+
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 5′-end-labeled (more ...) In vitro
nuclease assays were first performed in the absence or presence of 5 mM Mn2+
using recombinant, GST-tagged proteins and a 5′-end-labeled A30
RNA substrate (A). Protein preparations used are shown in Figure S2. Consistent with previous results (6
), WT Rrp44 (lane 2) exhibited strong 3′-exonucleolytic activity in the absence of Mn2+
, completely degrading the substrate to a 4 nt product. In contrast, the Rrp44-exo mutant showed little degradation activity (lane 3). However, in the presence of Mn2+
, both WT and mutant proteins cleaved the RNA, generating very similar degradation products (lanes 5 and 6). From these results, we concluded that Rrp44 harbors two distinct ribonuclease activities, which respond differently to Mn2+
ions under in vitro
conditions. The presence of Mn2+
inhibited 3′-exonuclease activity, whereas a second activity was stimulated.
To further characterize the novel activity, we performed in vitro nuclease assays using the exonucleolytically inactive Rrp44-exo mutant and 5′- and 3′-labeled A30 RNA substrates (B). Rrp44-exo generated a ladder of degradation intermediates from both substrates in the absence of added Mn2+ ions (lanes 2 and 6), likely due to co-purification of a low level of Mn2+ bound to the recombinant protein, but the degradation activity was strongly stimulated in the presence of added Mn2+ (lanes 4 and 8). The ability of the Rrp44-exo mutant to generate degradation intermediates from both 3′-labeled and 5′-labeled substrates shows that it either carries both 5′-exonuclease and 3′-exonuclease activities or, more likely, possesses an endonuclease activity.
To assess if this potential endonuclease activity was associated with the PIN domain of Rrp44, point mutations were introduced at each of the four conserved active-site amino acids (D91
N), to create the Rrp44-endo mutant. In addition, the Rrp44-exo mutation was combined with the four PIN-domain mutations (Rrp44-endo–exo) and with a point mutation in the S1 RNA-binding domain (Rrp44-exo-S1) (6
In vitro nuclease assays were performed using recombinant, GST-tagged proteins and a 5′-labeled substrate RNA derived from the 3′ region of the mouse 5.8S rRNA, which has a well defined, stable terminal stem structure (C and D). WT Rrp44 (C, lane 2) partially degraded the substrate to a 10 nt product. In contrast, Rrp44-exo generated a set of fragments with end-points in the loop-region of the substrate RNA (C, lane 3). These fragments were not generated by either the Rrp44-endo (C, lane 4) or Rrp44-endo–exo proteins (lane 5), indicating that their formation required a functional PIN domain. Rrp44-exo-S1 did generate the loop fragments, but lacked the major 10 nt product, suggesting that S1 RNA-binding activity might be more important for the exonuclease activity of Rrp44 than for endonuclease activity. A prominent product that is truncated by ~10 nt was detected in some samples. This appears to be due to a contaminating nuclease that is associated with purification of GST-fusion constructs from E. coli, as the same band was seen in analyses of other fusion proteins (data not shown). The truncated fragment appears to be very susceptible to degradation by the exonuclease activity of Rrp44, giving rise to the observed variations in signal. The PIN domain alone did not show in vitro activity. However, when purified from E. coli the PIN domain co-purified, apparently stoichiometrically, with the bacterial chaperone GroEL (indicated with an asterisk in B), strongly indicating that it is largely misfolded. We conclude that recombinant Rrp44 shows an endonuclease activity that requires Mn2+ ions and the metal-binding amino acids of the PIN domain.
The effects of the rrp44-endo mutation were also tested on viability. In the GAL::rrp44 strain, expression of Rrp44-endo clearly supported growth when WT Rrp44 was depleted by growth on glucose medium. In contrast, expression of Rrp44-endo–exo failed to support growth (data not shown), indicating that the endonuclease and exonuclease activities of Rrp44 share some redundant essential function. In plasmid shuffle experiments, expression of Rrp44-endo, but not Rrp44-endo–exo, supported viability in an rrp44Δ rrp6Δ double mutant strain (data not shown, but see B, lane 6). A low number of rrp44Δ strains complemented by the plasmid expressing Rrp44-endo–exo could be isolated by plasmid shuffle (data not shown). Rare cells are therefore able to adapt to loss of both the endonuclease and exonuclease activities of Rrp44 by epigenetic or physiological mechanisms.
Northern analyses were performed on the GAL::rrp44 strain expressing Rrp44-endo and Rrp44-endo–exo during Rrp44 depletion (A). These showed that the 5′-extended forms of the 5.8S rRNA and truncated forms of the 5′-ETS observed in strains expressing Rrp44-exo were suppressed in the rrp44-endo strain and altered in the rrp44-endo–exo strain. In particular, 5′-ETS fragments accumulating in the rrp44-exo strain (A, lane 4) were clearly reduced in strains expressing Rrp44-endo–exo (A, lane 8). This suggested that the endonuclease activity of Rrp44 is involved in formation of intermediate species that are stabilized by loss of Rrp44 3′-exonuclease activity. The longer form of the 5.8S + 30 RNA seen in the rrp6Δ strain expressing Rrp44-exo (B, lane 5) was not observed in the rrp6Δ strain expressing Rrp44-endo (B, lane 6). Accumulation of the truncated form of the 5S rRNA, seen in rrp44-exo strains, was not suppressed in the rrp44-endo–exo strain. However, the truncated 5S was accumulated in rrp6Δ strains, even in the presence of the rrp44-endo mutation.
Expression of only Rrp44 lacking both the endonuclease and exonuclease activity resulted in a phenotype that closely resembled that seen on depletion of Rrp44 (vector control in A, lane 10). Together the data indicate that the PIN domain of Rrp44 exhibits endonuclease activity in vitro and in vivo.
The PIN domain links Rrp44 to the core exosome
The archaeal exosome lacks a homologue of Rrp44, and yeast Rrp44 can be removed from the remaining nine components of the exosome by washing with high salt. This indicates that Rrp44 associates with a highly stable, nine component exosome core structure. A two-hybrid interaction was detected between the PIN domain of human Rrp44 and the RNase PH-homologue OIP2 (the human homologue of yeast Rrp43) (39
) suggesting that the PIN domain might be involved in protein–protein interactions that tether Rrp44 to the core structure. This model would be consistent with the absence of PIN domains from E. coli
RNase R and II (A), which function as monomers.
The association of Rrp44 with the exosome core was initially tested by glycerol gradient centrifugation of yeast cell lysates (A). WT, protein A-tagged Rrp44 largely co-sedimented with the core exosome component Rrp43 in ~14S complexes (fractions 14–17), with a small peak corresponding to the position of free Rrp44 (fractions 7–8). In contrast, protein A-tagged Rrp44 lacking the PIN-domain (Δ aa 86–203), was expressed at levels substantially below that of WT Rrp44, but predominantly sedimented as free protein. In Rrp44-depleted cells, Rrp43 becomes enriched in ~60S gradient fractions, reflecting stabilized exosome association with pre-ribosomes (L. Milligan and D.T., unpublished results), giving rise to the strong signal in the pellet fractions on the gradient shown.
Binding of Rrp44 to the core exosome was also assessed by in vitro binding assays using recombinant GST-tagged Rrp44 constructs expressed in E. coli (B) and core exosome purified from yeast, with or without removal of Rrp44 by washing with 800 mM MgCl2 (C). The GST-tagged constructs were bound to glutathione sepharose and incubated with the exosome preparations. The association of the exosome with the GST-fusion proteins was assessed by western blotting using antibodies specific for Rrp43 (D) and Rrp4 (data not shown). Exosome binding to the full-length Rrp44 (FL) (D, lane 2) was substantially stronger than to Rrp44 lacking the N-terminal PIN domain (ΔPIN) (D, lane 4), which was not above the GST background (D, lane 6). Notably, exosome binding was not affected by four individual mutations in the PIN domain (Rrp44-endo, see ) (D, lane 3). The PIN domain alone (PIN) (D, lane 5) gave a lower signal, but this clearly was above the GST background. As noted above, the low signal may reflect poor folding of the isolated PIN domain, as suggested by its copurification with bacterial GroEL. The signal from the Rrp44-depleted exosome (exosome –44) was stronger than for exosome purified at lower salt concentrations (exosome +44). However, some binding to Rrp44 was seen even without the high-salt wash, suggesting that Rrp44 is substoichiometric in the purified exosome.
We conclude that the PIN domain of Rrp44 plays a dual role in that it harbors endonuclease activity and also functions in tethering Rrp44 to the remaining nine subunit core of the exosome.