Assembly of Erb1, Nop7, and Ytm1 into Preribosomes Is Interdependent
Do individual components of the heterotrimeric Nop7-subcomplex influence the association of the other two proteins with preribosomes? To address this question, we assayed the constituents of preribosomes when Nop7, Erb1, or Ytm1 was depleted. Strains that express each of the three proteins under the control of the galactose-inducible and glucose-repressible GAL
promoter were constructed. Western blotting of proteins from whole cell extracts confirmed Nop7, Erb1, or Ytm1 were depleted to very low or undetectable levels in the respective strains upon shifting each strain from galactose- to glucose-containing medium (A). Consequently, growth of the cells slowed significantly (Pestov et al., 2001
; Adams et al., 2002
; Miles et al., 2005
). Preribosomal particles were affinity-purified from these strains using TAP-tagged Rpf2, which is present in 90S preribosomes and each of the consecutive 66S precursors to mature 60S ribosomal subunits (Zhang et al., 2007
). Rpf2 is required for a different pre-rRNA processing step than the Nop7-subcomplex (Zhang et al., 2007
); thus it is unlikely to directly interact with the Nop7-subcomplex. SDS-PAGE and silver staining of proteins from preribosomes purified from the undepleted and depleted strains revealed that although a specific subset of proteins was absent, pre-rRNPs were otherwise largely intact upon depletion of Nop7, Ytm1, or Erb1 (B). When either Nop7 or Erb1 was depleted, the other two components of the Nop7-subcomplex were completely or almost completely undetectable in preribosomes. In contrast, when Ytm1 was depleted, amounts of Nop7 or Erb1 in preribosomes were reduced to a lesser extent (B). These results are consistent with our previous findings that Ytm1 assembles into preribosomes after Nop7 and Erb1 (Miles et al., 2005
). The decreased levels of Nop7 and Erb1 in preribosomes from Ytm1-depleted cells indicate that assembly of Ytm1 into preribosomal particles is important for the retention of these two earlier assembled proteins. Because preribosomes undergo multiple consecutive rearrangements during their maturation, it seems very likely that dynamic intermolecular interactions would play an important role to maintain association of factors with particles. To assess whether the inability of Nop7, Erb1, or Ytm1 to associate with preribosomes upon depletion of one is due to instability of those proteins in cells, Western blots from whole cell lysates were carried out. On depletion of either Erb1 or Ytm1, wild-type levels of the other two proteins were detected in whole cell extracts, indicating no effects on their stability (A). Thus their absence from preribosomes was not simply caused by their absence from cells. In contrast, amounts of Erb1 and Ytm1 in whole cell extracts were substantially decreased upon depletion of Nop7 (A), indicating that their inability to associate with preribosomes upon depletion of Nop7 arises due to their instability in the absence of Nop7. We conclude that association of Erb1, Nop7, and Ytm1 with pre-RNPs occurs in an interdependent manner, whereas association of most other proteins with preribosomes is not affected in the absence of these three proteins.
Figure 1. Interdependence of assembly of Erb1, Nop7, and Ytm1 into preribosomes. Yeast strains containing GAL-NOP7, GAL-ERB1, or GAL-YTM1 were grown at 30°C in galactose medium to 3·107 cells/ml. A second culture of each strain was grown in galactose (more ...)
Ytm1 Binds Erb1 via Its C-terminal WD40 Repeats
Previously, we showed that Ytm1 interacts directly with Erb1 (Miles et al., 2005
). To determine which portion of Ytm1 is responsible for this interaction, we assayed binding in vitro to GST-Erb1 of the N-terminal 98 amino acids of Ytm1 (Ytm1-N) containing the Notchless-like element (Nal et al., 2002
), and the C-terminal 362 amino acids of Ytm1 (Ytm1-C) containing seven WD40 repeats (Miles et al., 2005
; A). Although the function of the Notchless-like element is unknown (see Discussion
), the WD40 repeats form a beta-propeller structure, a well-characterized protein–protein interaction domain (reviewed in Smith et al., 1999
S]methionine labeled Ytm1, Ytm1-N, and Ytm1-C were synthesized in vitro and assayed for binding to purified GST-Erb1. Ytm1-C bound to GST-Erb1 almost as well as full-length Ytm1, whereas Ytm1-N did not bind to GST-Erb1 (B). Therefore, Ytm1 binds Erb1 via its C-terminal WD40 domain.
Figure 2. The C-terminal WD-40 domain of Ytm1 (i.e., Ytm1-C) interacts directly with Erb1. (A) Schematic representation of Ytm1 and truncation constructs. Shown are the notchless-like element (NLE, white rectangle), and WD40 repeats 1–7 (hatched boxes). (more ...)
Binding to Erb1 Enables Ytm1-C to Assemble Into 66S Preribosomes
We then asked whether binding to Erb1 is required for association of Ytm1 with assembling ribosomes. If this is true, Ytm1-C should copurify with preribosomal particles, whereas Ytm1-N should not. To test this prediction, we expressed HA-tagged Ytm1, Ytm1-N, or Ytm1-C under control of the GAL promoter in cells also expressing endogenous Ytm1. Western blotting indicated that each protein was expressed at similar levels, 4 h after induction of the GAL constructs (Supplementary Figure S1A). Pre-rRNPs from cells expressing HA-tagged Ytm1, Ytm1-N, or Ytm1-C were purified using TAP-tagged Rpf2. Consistent with our prediction, Ytm1-C, but not Ytm1-N, associated with preribosomal particles to the same extent as full-length HA-tagged Ytm1 (A). Indirect immunofluorescence microscopy showed that both truncated Ytm1 proteins localized to the nucleus (Supplementary Figure S2A). Thus, the absence of Ytm1-N from preribosomes is unlikely to result from its inability to enter the nucleus.
Figure 3. Ytm1-C assembles into preribosomes and the Nop7-subcomplex in vivo. Yeast strains containing each of the GAL-inducible full-length or truncated YTM1 genes were grown at 30°C in synthetic medium containing 1% raffinose to ~1.5·10 (more ...)
To verify the interaction between Ytm1-C and Erb1 in vivo, TAP-tagged Ytm1-C was used to affinity-purify Ytm1-C and associated molecules from whole cell extracts. Significant amounts of both Erb1 and Nop7 copurified with Ytm1-C (B), indicating that Ytm1-C forms a heterotrimeric subcomplex with Erb1 and Nop7 in vivo. In contrast, neither Erb1 nor Nop7 copurified with TAP-tagged Ytm1-N (B). Western blotting using antibodies against other ribosome assembly factors showed that preribosomes copurified with Ytm1-C, but not Ytm1-N (B, and Supplementary Figure S3), providing independent evidence that Ytm1-C can assemble into preribosomal particles.
Previously we showed that the Ytm1–1 mutant protein contains two mutations, G398D and S442N, in the sixth and seventh WD40 repeats, which disrupt the interaction between Ytm1 and Erb1 and weaken the association of Ytm1 with preribosomes (Miles et al., 2005
). To confirm that binding to Erb1 enables Ytm1-C to assemble into preribosomes, we tested whether these mutations in Ytm1-1-C that decrease binding to Erb1 affect the association of Ytm1-1-C with pre-RNPs. We constructed strains expressing Ytm1–1 or Ytm1-1-C, each of which contains these two mutations (A). Although Ytm1-1-C was expressed at levels similar to Ytm1-C (Supplementary Figure S1A) and was primarily localized to the nucleus (Supplementary Figure S2A), it did not copurify with preribosomes (A).
Overexpression of Ytm1-C Has Dominant Negative Effects on Growth and Ribosome Biogenesis
The above results indicate that Ytm1-C might compete with wild-type Ytm1 for binding to Erb1 and assembly into preribosomal particles. However, the absence of the N-terminal 98 amino acids may prevent the truncated Ytm1-C protein from carrying out all necessary functions of full-length Ytm1. Thus preribosomes containing Ytm1-C might not undergo proper maturation. Alternatively, the truncated Ytm1-C protein might fold into an aberrant structure (“poison subunit”) and interfere with ribosome biogenesis (Herskowitz, 1987
). Consistent with these ideas, overexpression of Ytm1-C had a dominant negative effect on growth, whereas overexpression of either full-length Ytm1 or Ytm1-N did not affect growth rate (A). To determine the effect of overexpression of Ytm1-C on ribosome biogenesis, we assayed levels of ribosomal subunits by sucrose gradient sedimentation. Cells overexpressing either full-length Ytm1 or Ytm1-N contained wild-type levels of 60S subunits (C). In contrast, yeast overexpressing Ytm1-C contained significantly fewer free 60S subunits (C), indicating a defect in 60S ribosomal subunit assembly almost as strong as observed upon depletion or inactivation of Ytm1 (Harnpicharnchai et al., 2001
; Miles et al., 2005
). To further investigate defects in 60S subunit biogenesis in strains overexpressing Ytm1-C, we assayed the relative amounts of pre-rRNA processing intermediates and mature rRNAs. Surprisingly, we observed no significant differences in the relative amounts of pre-rRNA or mature rRNA in these cells compared with wild-type cells (E). These results suggest that overexpression of Ytm1-C must cause a defect in production of 60S ribosomal subunits that does not include perturbations of pre-rRNA processing.
Figure 4. Expression of Ytm1-C causes dominant negative effects on growth and ribosome biogenesis. (A) Wild-type cells or yeast expressing endogenous Ytm1 plus full-length or truncated Ytm1 proteins were grown to early log phase, serially diluted (10-, 100-, 1000 (more ...)
If Ytm1-C binds to Erb1 and competes with full-length Ytm1 to assemble into preribosomes, then disrupting the interaction between Ytm1-C and Erb1 should suppress the dominant negative phenotypes caused by Ytm1-C. Consistent with this idea, overexpression of the mutant Ytm1-1-C protein did not affect either growth or ribosome biogenesis (, A and C). In addition, when full-length Ytm1 was co-overexpressed together with Ytm1-C, the growth defect caused by Ytm1-C was suppressed (B).
All of these findings are consistent with our previous results that Ytm1 assembles into preribosomal particles later than Erb1 (Miles et al., 2005
) and that Erb1 is required for Ytm1 recruitment (). Additionally, our findings help explain why overexpression of WDR12 ΔNle, a similarly truncated version of WDR12, the mammalian homologue of Ytm1, causes dominant negative effects on cell proliferation and ribosome biogenesis in cultured mammalian cell lines (Hölzel et al., 2005
The N-terminal Conserved Region of Erb1 Interacts with Nop7 and Ytm1
Like Ytm1, Erb1 also contains seven WD40 repeats (Supplementary Figure S4; D. Wilson, personal communication). By binding directly to both Nop7 and Ytm1, Erb1 plays a central role in the formation of the Nop7-subcomplex (Miles et al., 2005
). However, which domain of Erb1 (or its mammalian homologue Bop1) is required for these interactions has not been determined. To systematically investigate domains involved in the interaction between Erb1 and other components in the Nop7-subcomplex and domains required for the recruitment of Erb1 into preribosomes, full-length Erb1 and a panel of four different truncated Erb1 constructs (A) were expressed from the GAL
promoter in cells also expressing endogenous full-length Erb1. Erb1-N contains the N-terminal 419 amino acids of Erb1, which includes a highly conserved region of unknown function (Pestov et al., 2001
). Erb1-C1 contains the C-terminal 388 amino acids of Erb1, which includes the seven WD40 motifs. Erb1-C2 contains amino acids 265–807, including half of the N-terminal conserved region and all of the WD40 repeats. Erb1-M, containing amino acids 383–639 (A), was identified as a ligand of Ytm1 in a genome-wide two-hybrid screen (data not shown). Western blotting indicated that all four truncated constructs were stably expressed (Supplementary Figure S1B). Indirect immunofluorescence microscopy showed that all four truncated proteins were present in both the cytoplasm and the nucleus. Among them, Erb1-C2 accumulated most strongly in the nucleolus (Supplementary Figure S2B).
Figure 5. The N-terminal conserved region of Erb1 interacts with both Nop7 and Ytm1 and assembles into preribosomes. (A) Schematic representation of full-length and truncated Erb1 constructs. Shown are the N-terminal conserved region of Erb1 (white rectangle) and (more ...)
We first tested which of these Erb1 fragments is present in preribosomal particles. Pre-rRNPs were purified from cells expressing each of the truncated Erb1 constructs, using TAP-tagged Rpf2. Western blotting showed that Erb1-N and Erb1-C2 assemble into preribosomes at levels similar to full-length Erb1, whereas Erb1-C1 and Erb1-M do not (B). Thus, amino acids 265–383, which are present in both Erb1-N and Erb1-C2, but not in Erb1-C1 and Erb1-M (A), are important for recruitment of Erb1 into pre-rRNPs.
To identify whether Nop7, Ytm1, or any other molecules associate with each domain of Erb1 in vivo, we carried out affinity purification with each TAP-tagged truncated Erb1 protein. Nop7 and Ytm1, as well as small amounts of other preribosomal proteins (Supplementary Figure S3), copurified with Erb1-N-TAP and with Erb1-C2-TAP (C). This confirmed our previous findings that both Erb1-N and Erb1-C2 assemble into preribosomes. No Nop7 and very little Ytm1 copurified with Erb1-C1-TAP (C). Consistent with the two-hybrid interaction observed between Ytm1 and Erb1-M (data not shown), Ytm1, but not Nop7, copurified with TAP-tagged Erb1-M (C). Erb1-M contains only three WD40-repeats (amino acids 420–639), which may not be able to fold into a β-propeller structure and serve as a stable functional interaction domain. Thus we conclude that the N-terminal region of Erb1-M lacking the WD40 domains (amino acids 383–419) most likely interacts with Ytm1. Other protein(s) might contribute to the interaction between each truncated Erb1 and these ligand(s), but this seems unlikely because both Nop7 and Ytm1 bind to Erb1 directly in vitro (Miles et al., 2005
). Additionally, interactions between Erb1-N and Ytm1 or Nop7 were confirmed by yeast two-hybrid assays (data not shown).
Therefore, these results indicate the following: 1) amino acids 265–383, which are present in both Erb1-N and Erb1-C2, but not in Erb1-M, are required for interaction with Nop7, and are important for Erb1 to assemble into preribosomes, 2) the central region of Erb1 containing amino acids 383–419 is involved in interactions between Erb1 and Ytm1 (see ), and ) interactions with both Nop7 and Ytm1 enable Erb1-N and Erb1-C2 to stably associate with preribosomal particles. Conversely, the inability to bind to Nop7 most likely prevents Erb1-C1 and Erb1-M from assembling into or remaining associated with pre-rRNPs. These results are consistent with the findings that efficient association of Erb1 with preribosomes is dependent upon both Nop7 and Ytm1 (B).
(A) Interaction domains within the Nop7-subcomplex. Light pink or light green boxes in each protein indicate the smallest region shown to interact with their full-length ligands. (B) Architecture of the Nop7-subcomplex.
Effects of Overexpression of Erb1 Truncations on Growth and Ribosome Biogenesis
We also investigated effects of overexpression of Erb1 truncations on growth rate and ribosome biogenesis. As predicted, overexpression of Erb1-C1 and Erb1-M, two Erb1 truncations that cannot associate with preribosomes, did not cause any defects in growth (A) or ribosome biogenesis (B). Similarly, overexpression of full-length Erb1 did not affect cell growth or ribosome assembly (, A and B). This result is different from two previous observations, which showed that overexpressing Bop1 in cultured mouse or human cells has mild or strong dominant negative effects on cell proliferation and pre-rRNA processing (Strezoska et al., 2000
; Rohrmoser et al., 2007
). The differences between these results in mouse and human cells and yeast may indicate subtle structural, functional, and regulatory changes during evolution.
Figure 6. Expression of Erb1-C2 causes dominant negative effects on growth and ribosome biogenesis. (A) Wild-type cells or yeast expressing endogenous Erb1 plus full-length or truncated Erb1 proteins were grown to early log phase and serially diluted as in (more ...)
In contrast, overexpression of Erb1-C2 had a dominant negative effect on growth (A) and on levels of 60S ribosomal subunits (B), indicating a defect in maturation of 66S preribosomes. Primer extension and Northern blotting experiments showed that in the strains overexpressing Erb1-C2, the relative amounts of the 27SA3
pre-rRNA intermediate increased, whereas 27SB and 7S pre-rRNAs, as well as 25S and 5.8S rRNAs, decreased (C). These pre-rRNA processing defects are similar to those in the Erb1 depletion strain, but are not as strong (Pestov et al., 2001
; C). These results with Erb1-C2 suggest that the N-terminal 264 amino acids of Erb1, missing from Erb1-C2, are important for ribosome biogenesis and pre-rRNA processing, or else that Erb1-C2 might function as a “poison subunit” to interfere with assembly of 60S ribosomal subunits. Consistent with these results, overexpression of Bop1Δ, a mammalian Erb1 deletion construct containing the same domains as Erb1-C2, causes dominant negative effects on cell proliferation and 60S ribosomal subunit assembly (Strezoska et al., 2000
Surprisingly, although Erb1-N also binds to Nop7 and Ytm1 and assembles into pre-rRNPs, overexpression of Erb1-N does not cause dominant negative effects on growth, 60S ribosomal subunit assembly, or pre-rRNA processing (, A–C).
The WD40 Motifs of Erb1 Are Not Essential
The absence of effects on ribosome biogenesis caused by overexpression of Erb1-N, despite the significant association of this truncated protein with preribosomes, suggests that Erb1-N is sufficient to perform all essential functions of Erb1. To test this hypothesis, Erb1-N was expressed from its own promoter in a strain that also contains a GAL-ERB1gene. Thus, when cells grown in galactose are shifted to glucose, Erb1-N but not full-length Erb1 will be expressed. Consistent with our hypothesis, expression of ERB1-N rescued the lethal phenotype of shutting off GAL-ERB1 (A). In contrast, neither Erb1-C1 nor Erb1-C2 could complement the lethality caused by depletion of wild-type Erb1 (A), consistent with our observations that Erb1-C1 cannot assemble into preribosomes and that Erb1-C2 can assemble into preribosomal particles, but causes dominant negative effects. Compared with wild-type cells expressing full-length Erb1, cells expressing only the truncated Erb1-N protein showed no obvious defects in 60S ribosomal subunit assembly and polysome formation (B). Consistent with this lack of noticeable effects on ribosome production or function, the relative levels of 27SA3 or 27SB pre-rRNAs in cells expressing Erb1-N were only slightly different from those in wild-type cells (C). Mild defects in ribosome assembly in vivo are sometimes exacerbated by growing cells under less than ideal conditions, such as high or low temperatures. However, cells expressing only Erb1-N exhibited no growth defects at 37 or 13°C, compared with 30°C (data not shown). Nevertheless, the reproducible mild defects in pre-rRNA processing in cells expressing Erb1-N leave open the possibility that the conserved C-terminal WD40 domain of Erb1 may be involved in some function of Erb1 in ribosome biogenesis.
Figure 7. The C-terminal WD40 domain of Erb1 is not essential for growth or ribosome assembly. (A) Empty vector or a plasmid containing each ERB1 truncation driven by the ERB1 promoter was introduced into the GAL-ERB1 strain. Cells were grown to early log phase (more ...)
The Central Region of Nop7 (Nop7-M) Binds to Erb1, Assembles into 66S Preribosomal Particles, and Causes Mild Dominant Negative Effects
Previously, we showed that Nop7 interacts directly with Erb1 but not Ytm1 in vitro (Miles et al., 2005
). Wild-type Nop7 and a panel of Nop7 truncations were generated to investigate domains involved in interaction between Nop7 and Erb1 and in recruitment of Nop7 into preribosomes (A). The N-terminal half of Nop7 (Nop7-N) contains a highly conserved pescadillo-like sequence of unknown function, and the C-terminal half (Nop7-C) contains three predicted protein–protein interaction domains: a BRCT motif (Clapperton et al., 2004
) and two coiled-coil domains (A). Nop7-M lacks the amino-terminal 51 amino acids and the carboxyl-terminal 120 amino acids of wild-type Nop7, but contains most of the amino-terminal pescadillo-like region, the BRCT domain, and one coiled-coil domain (A). HA-tagged full-length Nop7 and these three HA-tagged Nop7 truncations were expressed from the GAL
promoter in cells also expressing endogenous full-length Nop7 (Supplementary Figure S1C). To identify the domains of Nop7 required for its recruitment into preribosomes, pre-rRNPs were purified from cells expressing each truncated Nop7 construct, using TAP-tagged Rpf2. Western blot analysis demonstrated that Nop7-M, but not Nop7-N or Nop7-C, was present in purified preribosomal particles at levels similar to full-length Nop7 (B). Indirect immunofluorescence microscopy showed that each of the truncated Nop7 proteins was localized to the nucleus (Supplementary Figure S2C). Thus, the absence of Nop7-N and Nop7-C from preribosomes is unlikely to result from their inability to enter the nucleus. When each truncated Nop7 construct was TAP-tagged and used for affinity purification, more Erb1 and Ytm1 copurified with Nop7-M than with Nop7-N or Nop7-C (C), indicating domains present only in Nop7-N or Nop7-C are not sufficient for Nop7 to bind Erb1 stably. Although the interaction between Erb1 and each Nop7 truncation could be indirect, we believe that these interactions are direct, because Nop7 and Erb1 bind to each other directly in vitro (Miles et al., 2005
). We also observed that overexpression of Nop7-M caused mild defects in growth (D) and ribosome biogenesis (E). Consistent with these mild defects in cells overexpressing Nop7-M, only small amounts of 27SA3
pre-rRNA accumulated relative to 27SB pre-rRNAs (F). In contrast, overexpression of full-length Nop7, Nop7-N, or Nop7-C did not affect growth or ribosome assembly (, D and E). Taken together, these results indicate that Nop7 binds to Erb1 via amino acids 52–484 containing multiple potential protein–protein interaction motifs. The mild dominant phenotype of Nop7-M suggests that either the amino- or carboxyl-terminal portion of Nop7, or both, missing from Nop7-M, might be important for ribosome biogenesis.
Figure 8. Interaction domains of Nop7. (A) Schematic representation of Nop7 and its truncation constructs. Shown are the N-terminal pescadillo domain of Nop7 (white rectangle), BRCT domain (dotted box), and two coil-coiled domains (gray boxes). (B) Pre-rRNPs were (more ...)
Previous studies on Pes1, the mammalian homologue of Nop7, are consistent with and support our results. Transposon-based insertion mutagenesis of Pes1 suggested that a stretch of seven amino acids located in the pescadillo-like domain is involved in interaction of Pes1 with Bop1 and that this interaction is important for Pes1 to function in pre-rRNA processing and cell proliferation (Lapik et al., 2004
). Systematic deletions of Pes1 and mutation of the BRCT domain in Pes1 indicated that the BRCT domain, a coiled-coil domain, and most of the N-terminal pescadillo-like protein domains are important for incorporation of Pes1 into the PeBoW-complex (Grimm et al., 2006
; Hölzel et al., 2006