Our working hypothesis is that the numerous assembly factors associate with nascent ribosomes in a hierarchical and cooperative manner, through protein–protein or protein–RNA interactions, to drive forward the formation of consecutive assembly intermediates. Therefore, our approach to begin to uncover the precise roles of these factors is to systematically investigate physical or functional interactions among them.
Stable interactions among some assembly factors and r-proteins are evident by isolating assembly subcomplexes containing them (25
). These subcomplexes might serve as smaller building blocks to minimize the complexity of assembly of these enormous ribonucleoprotein particles containing so many components. Importantly, identifying and characterizing these physical clusters of assembly factors will enable discovery of cofactors and substrates of these factors, thus unraveling their functions, and will also help define the local topology within pre-rRNPs.
Rearrangements of local structures within assembling ribosomes may be propagated by allosteric mechanisms to alter the global architecture of pre-rRNPs. Such reorganizations might affect recruitment or activity of assembly factors working in the same step of subunit biogenesis, even though these proteins may not physically interact. Thus, to obtain a comprehensive picture of ribosome assembly, it is necessary to ‘walk out’ of neighborhoods defined by physical subcomplexes within pre-ribosomes and to investigate interactions among all assembly factors that function together in one specific step of pre-rRNA processing and assembly. Studying molecules in one ‘functional cluster’ will help to understand how these molecules coordinate their precise functions with each other in one assembly step.
We previously discovered that six assembly factors required for processing of 27SA3
pre-rRNA assemble early into pre-ribosomes, are mutually interdependent for association with pre-ribosomes and are important for assembly of r-proteins with domain I and II of 25S/5.8S rRNAs (31
) (Jakovljevic et al
., accepted with minor revisions). Here, we have studied recruitment of the factors required for the next step in pre-rRNA processing, removal of ITS2 from 27SB pre-rRNA. Previous experiments to map the relative timing of association of B-factors with pre-ribosomes demonstrated that seven of these proteins assembled rather early, with 90S pre-ribosomes or 66S particles containing 27SA2
). In contrast, Nsa2 and Nog2 assembled later, with 27SB pre-rRNA (17
). The entry points of Dbp10, Nip7 and Nop2 have not been studied. Experiments described in this article are consistent with and extend previous results to generate a higher resolution picture of the assembly hierarchy.
Building on work from the Fromont-Racine group, we began by assaying the association of the late-entering B-factors, Nsa2 and Nog2, in the absence of the other B-factors. We show that all of the B-factors are required to recruit the GTPase Nog2, but only a subset (Nop2/Nip7, Dbp10, Tif6, Rlp24 and Nog1) are required to recruit Nsa2. Fromont-Racine and coworkers showed that Nsa2 is necessary to recruit Nog2. Here, we show that in the absence of Rpf2 or Sbp4, Nsa2 is still recruited into pre-ribosomes, but Nog2 is not. Thus, Nsa2 alone is not sufficient to recruit the GTPase Nog2.
We then extended our model by systematically testing the interdependence of all the B-factors. We discovered that these B-factors are loaded into pre-ribosomes sequentially via two largely separate assembly routes that converge on Nog2 (C). Association of Nop2 and Nip7 with each other and with pre-ribosomes is necessary for stable association of downstream B-factors, beginning with the Rpf2 subcomplex (Rpf2, Rrs1, r-proteins L5 and L11 and 5S rRNA), and Rlp24, Tif6 and Nog1. Members of each of these three sets of proteins are mutually interdependent for assembly and exhibit significant physical and genetic interactions with each other (Supplementary Table S3
and A). Furthermore, many contain RNA binding motifs and thus are likely to bind pre-rRNA. Taken together, these results suggest that processing of 27SB pre-rRNA requires formation of one or more assembly platforms of B-factor subcomplexes within pre-ribosomes, several steps before the processing event. In contrast, the last steps in recruiting of the B-factors occur later in assembly, after formation of 27SB pre-rRNA.
Figure 9. Physical and genetic interactions provide clues about the location of the B-factors in pre-ribosomes. (A) Predicted locations of B-factors in assembling 60S subunits. Binding sites of proteins were predicted using a combination of genetic and physical (more ...)
In the absence of each B-factor, other B-factors associate less well with pre-ribosomes, but to varying extents. Western blotting showed that the B-factors are usually not completely absent from pre-ribosomes, but rather diminished. The strongest effect that we observed in our mutants was the association of Nsa2 and Nog2. This probably reflects blocking assembly at a step before these proteins associate with pre-ribosomes. Conversely, effects on the association of other B-factors were often not as strong. One possibility is that we observe small changes in the levels of these proteins because we purify a different population of assembly intermediates in cells depleted of B-factors. We believe that this is not the case because TAP-tagged Nop7 does not co-IP significantly different pre-rRNA intermediates in B-factor mutants (Supplementary Figure S1
and data not shown). In fact, Nop7 co-IPs slightly greater amounts of 27SB pre-rRNA in the absence of the B-factors. Unlike Nsa2 and Nog2, the other B-factors associate with 35S or 27SA2
containing pre-ribosomes and remain associated throughout the lifetime of 27SB pre-rRNA. Because 27SB-containing pre-ribosomes are the longest-lived intermediates and these intermediates accumulate in B-factor mutants, we predict that the small changes that we see might in fact be slightly underrepresented.
The experiments performed in this study fail to distinguish two possibilities that could occur in the absence of each B-factor: (i) The other B-factors fail to associate with pre-ribosomes. (ii) The other B-factors associate with pre-ribosomes, but fail to be stably incorporated. We believe the former to be true regarding Nsa2 and Nog2 and the later regarding the other B-factors. Each B-factor in our model (C) does not necessarily recruit the next through direct interaction, but rather may form a structure or niche in the pre-ribosome that allows stable incorporation of the next B-factor. Thus, each B-factor only stably associates with pre-ribosomes after stable incorporation of the preceding B-factors.
Our model shows that the B-factors associate with pre-ribosomes through two converging pathways that result in recruiting Nog2. Parallel assembly pathways that converge to a single intermediate have been observed in ribosome biogenesis and are proposed to be quality control mechanisms (53–55
). Johnson and coworkers (55
) showed that cytoplasmic release of assembly factors occurs via parallel pathways that converge to trigger the release of Tif6. This convergence of pathways may represent a quality control checkpoint to ensure Tif6 is not released before the assembling 60S subunit is competent for translation. Lamanna and Karbstein (56
) speculate that parallel assembly pathways may converge before irreversible steps in assembly. They reason that irreversible steps should be tightly regulated and it is easier to survey a single intermediate rather than a number of different parallel intermediates before proceeding to the next step of assembly. This may be reflected in the recruitment of Nog2 to pre-ribosomes. As the last known B-factor to associate with pre-ribosomes, Nog2 may be a key factor in triggering C2
cleavage. Recruitment of Nog2 by two parallel pathways may ensure that it is not loaded onto pre-ribosomes prematurely, thus preventing premature cleavage at the C2
Of the 12 assembly factors required for processing of 27SB pre-rRNA, 4 are thought to exert their action by using the power of NTP hydrolysis. Involvement of this many NTPases during this step suggests substantial rearrangements may occur. Consistent with this, 27SB pre-rRNA is one of the most abundant pre-rRNA intermediates destined for 60S subunits, with a lifetime substantially longer than other pre-rRNAs (57
). The early assembling protein Spb4, a DBP and potential RNA-dependent ATPase, and the late assembling factor Nsa2, are necessary to recruit the GTPase Nog2. Neither Spb4 nor Nsa2 alone is sufficient to recruit Nog2. Spb4 associates early with pre-ribosomes, whereas Nsa2 associates later. Temporal separation of the association of these two proteins may act as a proofreader of ribosome assembly, ensuring that Nog2 is recruited to the pre-ribosome only at the proper time. Because Sbp4 and Nog2 are at the end of the recruiting pathway (C), it is tempting to speculate that removal of ITS2 is triggered by the concerted action of these NTPases. Spb4 might be regulated by Nog2, analogous to regulation of the spliceosomal ATPase Brr2 by the GTPase Snu114 (58
). During spliceosome assembly, GTP-bound Snu114 activates Brr2, whereas GDP-bound Snu114 represses Brr2 function. Alternatively, Nog2 alone could harness the energy of GTP hydrolysis to cause a conformational switch within the pre-ribosome. In particular, the A3
assembly factors Nop15 and Cic1 that bind to ITS2 may need to be removed or reorganized (59
). GTP hydrolysis may cause a structural change within ITS2 that makes binding of Nop15 and Cic1 less favorable in the new conformation (60
). Consistent with Nog2 being a member of the myosin/kinesin superfamily of GTPases, Nog2 could also act in a mechanical fashion, using GTP hydrolysis to physically displace Cic1 or Nop15 (60
Rearrangements before and during processing of 27SB pre-rRNA may also occur as a result of posttranslational modification of assembly factors. We show that the B-factors Rpf2, Spb4, Nog1 and Nog2 are specifically reduced in pre-ribosomes when the protein kinase TOR is inactivated. On the basis of our recruiting model, we would predict that if Nog1 was diminished in pre-ribosomes, Nsa2 would also be diminished. Contrary to this idea, we do not see a change in Nsa2 on inactivation of TOR signaling. This may indicate that these proteins are not failing to be recruited to pre-ribosomes, but rather are recruited normally, but then prematurely dissociate from pre-ribosomes on inactivation of TOR signaling. Interestingly, three of these B-factors are the putative NTPases Spb4, Nog1 and Nog2. These NTPases may fail to stably assemble into pre-ribosomes in the absence of posttranslational modifications. This could be analogous to r-protein S3, which must undergo a series of phosphorylation and dephosphorylation events to stably associate with pre-40S intermediates and promote formation of the 40S beak (62
Alternatively, inactivation of TOR signaling may repress expression of a subset of assembly factors. Nog1 was previously shown to be regulated by TOR kinase (43
). Inactivation of TOR caused decreased transcription of NOG1
. It was also shown that Nog1 is rapidly turned over, although independently of TOR signaling. We show that on inactivation of TOR, levels of Rpf2, Spb4, Nog1 and Nog2 are diminished in whole-cell extracts. Expression of the genes encoding these proteins, as well as other assembly factors and r-proteins, is controlled and coordinated by a set of core promoter sequences termed the Ribi
ogenesis) and RP regulon. In addition, inactivation of TOR signaling represses expression of genes controlled by the Ribi
and RP regulons (45
). Thus, Rpf2, Spb4 and Nog2 may be rapidly turned over, similarly to Nog1.
A current challenge in understanding ribosome biogenesis is determining where and how assembly factors contact the pre-ribosome. Recent studies on crosslink proteins to RNA with UV light have revealed the rRNA-binding sites of some assembly factors (59
). In addition, advances in cryo-EM have provided information about the locations of assembly factors in pre-ribosomes (38
), and higher resolution crystal structures of eukaryotic ribosomes are allowing better visualization of how r-proteins contact rRNA (74
). Although these techniques are powerful for determining how and where assembly factors contact the pre-ribosome, their application is limited in ribosome assembly. For example, not all assembly factors contact rRNA and thus cannot be cross-linked to determine their binding sites. Of those proteins that do bind RNA, crosslinking may not capture all RNA-binding sites in these dynamic particles. In addition, structural analyses often require a fairly homogenous purified sample. Because most assembly factors that function in 60S subunit biogenesis are present in more than one consecutive intermediate, obtaining homogenous samples for cryo-EM or generating crystals is difficult. Therefore, other methods need to be employed to help predict and determine the binding sites of assembly factors.
We used known genetic and physical interactions among the B-factors to generate a model of the locations of some of these assembly factors (A and Supplementary Table S3
). Nop2 and Nip7 form a subcomplex and are synthetically lethal with deletion of genes encoding r-proteins L26B or L17A, respectively (75
). These two r-proteins are adjacent to each other in domain I of 25S/5.8S rRNA, lying near the polypeptide exit tunnel (74
). Consistent with the synthetic lethal interactions observed between these two r-proteins and the B-factors Nop2 and Nip7, L17 and L26 function in processing of 27SB pre-rRNA (M. Gamalinda, personal communication). This suggests that Nop2 and Nip7 might dock pre-ribosomes near these two r-proteins, close to the exit tunnel (A).
Cryo-EM studies show that Tif6 binds L23 and L24 on the intersubunit side of assembling 60 subunits and functions to prevent premature association of the 60S and 40S subunits (38
). Tif6 is one of the last assembly factors to be released from the assembling ribosome. Its removal is dependent on the Shwachman-Bodian-Diamond syndrome protein homologue Sdo1 and the GTPase Efl1 (40
). Sdo1 was shown to interact with r-protein L3 in a two-hybrid screen, positioning Sdo1 near Tif6. Interestingly, Sdo1 was also shown to bind directly to Nip7 (76
). Thus, there may be a physical connection between Nip7, Sdo1 and Tif6. This provides a testable model that Sdo1 may be involved in releasing not only Tif6 but also other B-factors.
L5, L11 and 5S rRNA, which are members of the Rpf2 subcomplex, form the central protuberance (CP) near the top of 60S subunits (74
). Thus, it is likely that when Rpf2 and Rrs1 deliver L5, L11 and 5S rRNA to assembling ribosomes, they are located in a comparable position. We speculate that Rpf2 and Rrs1 are located on the intersubunit side of the CP based on the following evidence: (i) Rrs1 was shown to interact with Ebp2 in both two-hybrid and protein complementation assay (PCA) screens (77
); (ii) Ebp2 exhibits two hybrid interactions with Brx1 (79
) and (iii) Brx1 interacts with Tif6 by PCA (78
). These three pieces of evidence suggest a chain of interactions beginning with Tif6, perpetuated through Brx1 and Ebp2, and ending with the Rpf2 subcomplex.
Nsa2 exhibits two-hybrid interactions with r-proteins L4, L15 and L18 (23
) (B), which are located adjacent to each other on the left edge of the 60S subunit when viewed from the intersubunit face. Nsa2 shares a number of both physical and genetic interactions with Rlp24, Nog1 and Mak11, suggesting that these proteins may associate with pre-ribosomes in close proximity to each other (21
). Consistent with this idea, our results, with those of Saveanu et al.
, show that Rlp24, Nog1 and Mak11 are interdependent for their assembly into pre-ribosomes and all three are required to recruit Nsa2. Interestingly, overexpression of the A3
factor Nop7 suppresses mutations in nog1
). Nop7 is a member of a group of mutually interdependent proteins required for processing 27SA3
). UV crosslinking has shown most of the A3
factors bind rRNA in domain I of 25S/5.8S rRNA or in ITS2, and in their absence, these regions are not properly assembled (31
). Consistent with genetic interactions between Nog1 and Nop7, in the absence Nop7 and mutually interdependent A3
factors, Nog1 and Rlp24 fail to associate with pre-ribosomes, suggesting that they may also bind near the A3
) (data not shown). Specifically, Nop7 was shown to bind rRNA near helix 54 in domain III (59
) (B). This region of rRNA is in close proximity to the rRNA-binding sites of L4, L15 and L18 further supporting the idea Rlp24, Nog1, Mak11 and Nsa2 bind the left edge of the 60S subunit. It is also interesting that inactivation of TOR signaling causes both Nog1 and Nop7 to associate less well with pre-ribosomes (43
), suggesting that this neighborhood could be a target of TOR regulation.
Although we were unable to predict the binding sites of all B-factors, our model provides a number of important insights. First, we predict that a large majority of the B-factors are binding on the intersubunit face. Could some of these factors prevent premature subunit joining analogous to Tif6? Could Nog2 remove or reorganize these proteins analogous to other GTPases that function on the subunit interface (55
)? Second, we predict that Rlp24, Nog1 and Nsa2 are binding near r-proteins L4, L15 and L18, respectively. These are in close proximity to the ITS2-proximal stem, where ITS2 would be predicted to be located in assembling 60S subunits. Our predictions for the location of these B-factors put them in prime position to facilitate the removal of ITS2.