Previously Ebp2 and Brx1 were identified as phylogenetically conserved nucleolar proteins involved in early steps of 60S ribosomal subunit assembly in yeast. In the ebp2-1
temperature-sensitive mutant, processing of 27SA2
pre-rRNA is perturbed (7
). Upon depletion of Brx1, 27SA2
pre-rRNAs accumulate and 27SBS
pre-rRNA is diminished (25
). Here, we have identified brx1
mutations that exhibit synthetic lethal interactions with the ebp2-14
mutation. Three of these four brx1
mutations also eliminate the strong two-hybrid interaction between Ebp2 and Brx1. Despite this inability to interact with each other, both mutant ebp2 and brx1 proteins can associate stably with pre-ribosomes. Nevertheless, these sl interactions result in defects in pre-rRNA processing. Thus, these interactions may enable us to investigate functions of Ebp2 and Brx1 after their initial docking into pre-rRNPs. We discuss a broader network of physical and functional interactions in which Ebp2 and Brx1 might participate to enable early steps of 60S subunit biogenesis.
We previously suggested that Ebp2 might enter 90S pre-ribosomal particles based on two-hybrid data demonstrating that Ebp2 associates with the 40S subunit assembly factors Utp11 and Faf1, and r protein S16 (9
). Consistent with Ebp2 and Brx1 functioning early in assembly, both proteins copurify with ribosome assembly factors that function at early or middle steps in the pathway (2
). Using the consecutive pre-rRNA processing intermediates as landmarks, we also found that Ebp2 and Brx1 are associated with the 35S pre-rRNA present in 90S pre-ribosomes, as well as each of the 27S pre-rRNAs found in 66S pre-rRNPs (B). Neither of these experimental approaches distinguishes the order in which Ebp2 and Brx1 dock with nascent particles. However, two observations suggest that Ebp2 might assemble before Brx1: (i) Brx1 depends on Ebp2 to associate with pre-ribosomes, but not vice versa (C, right). (ii) Depletion of Brx1 blocks 27SA3
pre-rRNA processing downstream of the step blocked by depletion of Ebp2 (A). Nevertheless, a higher resolution view of the assembly hierarchy awaits better tools to investigate ribosome biogenesis.
The genetic interactions between ebp2-14
could provide additional insight into the function of Ebp2 and Brx1. At 25°C, each of the single ebp2
missense mutants has no apparent defect in growth or in production of 60S ribosomal subunits (A, A, see ref.8). Even the brx1-227
mutant, which is temperature sensitive for growth, appears not to have a defect in production of 60S ribosomal subunits at 25°C (B). In contrast, the ebp2-14 brx1
double mutants are unable to grow, and are defective in production of 60S subunits and processing of 27SA2
pre-rRNAs (C, 4A). The ebp2-14
mutation together with brx1-52, brx1-102 or brx1-227
mutations prevents stable interaction of ebp2 and brx1 mutant proteins with each other in a two-hybrid assay (B). The combination of ebp2-14
has little effect on the interaction. As the combination of ebp2-12
(other allele, see ref. 8) and brx1-172
also has no effect on the interaction (data not shown), we speculate that the brx1-172 mutant protein, similar to Brx1, might interact with ebp2 mutant proteins as well as with Ebp2. Despite the loss of interaction, both ebp2-14
mutant proteins can efficiently assemble into nascent ribosomes in the double mutants, as well as the ebp2-14 brx1-172
mutant proteins where the two-hybrid interaction is not abolished (B). This result indicates that other protein–protein or protein–RNA interactions within assembling ribosomes must enable Ebp2 and Brx1 to stably associate with pre-ribosomes. A similar result for assembly factors Imp4 and Mpp10 was found previously by Gallagher and Baserga (26
). Mutations in Imp4 that disrupt two-hybrid interactions with Mpp10, also cause a slow growth phenotype and perturb pre-rRNA processing, but nevertheless do not prevent either protein from associating with the SSU processome.
Although the ebp2-14 and the brx1-102 mutant proteins can assemble into pre-ribosomes, the ebp2-14 brx1-102 double mutant is defective in processing of 27SA2 and 27SA3 pre-rRNAs, more so than either single mutant (B). Furthermore, the ebp2-14 brx1-172 double mutant in which the Ebp2–Brx1 interaction is not diminished, nevertheless produces fewer 60S subunits than either single mutant, and is defective in processing of 27SA3 as well as 27SA2 pre-rRNAs (B).
The effects on ribosome assembly upon depleting either Ebp2 or Brx1 support the existence of a broader network of pre-ribosomal interactions with Brx1 and Ebp2 (). Brx1 depends on Ebp2 to associate with early pre-ribosomes, but Ebp2 does not require Brx1 to do so (C, right). Thus, there must be other molecules in pre-rRNPs not affected by the absence of Brx1 that help anchor Ebp2 in pre-rRNPs. On the other hand, Brx1 may not depend on direct interactions with Ebp2 to assemble, but on a third molecule in pre-ribosomes, whose presence or anchoring function is diminished by the absence of Ebp2 ().
Figure 8. Model of dependence of Brx1 on Ebp2 for assembly into pre-ribosomes. Ebp2 (E) and Brx1 (B) are represented with yellow and green, respectively. X (red) and Y (blue) represent other proteins present in pre-ribosomes. Each X and each Y can represent one (more ...)
The best candidates for factors that help Ebp2 or Brx1 enter pre-ribosomes and function in these particles are four other ribosome assembly factors and r proteins found in subcomplexes together with Ebp2 or Brx1. Krogan et al.
) found that Brx1 copurified with Nop12 and Pwp1, when TAP-tagged Pwp1 was used for affinity purification from high speed supernatants of whole cell extracts from which intact pre-ribosomes and ribosomes were removed. By a similar approach, Zhang et al.
) found that Ebp2, Brx1, Nop12 and r proteins L8 and L15 copurified with TAP-tagged Pwp1. Consistent with these observations, Ebp2 interacts not only with Brx1 in two-hybrid assays, but also with Nop12 (A).
The association of Brx1 and Ebp2 with these four assembly factors and r proteins provides some hints about their possible location within pre-ribosomes. The r proteins L8 and L15 lie adjacent to each other in mature 60S ribosomal subunits, near the proximal stem formed by base-pairing of the 3′-end of 5.8S rRNA with the 5′-end of 25S rRNA (4
). During biogenesis of the 60S subunit, these 5′-and 3′-ends of rRNA are formed by processing of the internal transcribed spacer 2 (ITS2) that lies between them. Nop12 cross-links to nucleotides immediately adjacent to the proximal stem, but distal from ITS2 (29
). This physical neighborhood of six pre-ribosomal proteins, including Brx1 and Ebp2, is also functionally interconnected. The phenotype of nop12
mutants is similar to that of brx1
processing are perturbed (JJ and JT, unpublished).
The ‘A3 cluster’ of assembly factors required for processing of 27SA3
) may also be part of a larger local neighborhood of proteins that interact with Ebp2 and Brx1 and function together with them. Erb1 and Nop7 crosslink to rRNA in domains I and III near the proximal stem (29
), and Ytm1 binds directly to Erb1 (30
). Nop15 and Cic1 cross-link to sequences in ITS2, which lies adjacent to the proximal stem (29
). Thus, it seems likely that Ebp2 and Brx1 are located in pre-ribosomes near the proximal stem and function together with Nop12, Pwp1, r proteins L8 and L15, and perhaps other nearby A3
factors, to create RNP structures necessary for early steps of 27S pre-rRNA processing.
How might the genetic and physical interactions between Ebp2 and Brx1, and between these two proteins and the other pre-ribosomal proteins described above, relate to their function in ribosome assembly and pre-rRNA processing? Their overlapping mutant phenotypes indicate that these proteins participate either directly or indirectly in processing of 27SA2
pre-RNA and subsequently 27SA3
pre-rRNA. This first processing step involves endonucleolytic cleavage at the A3
site in the internal transcribed spacer 1 (ITS1) by the RNase MRP endonuclease (31–33
). Subsequently, 77
nt at the 5′-end of the resulting 27SA3
pre-rRNA are removed by the 5′–3′ exonucleases Rat1, Xrn1 and Rrp17, stopping precisely at the B1S
site, to produce 27SBS
). In both steps, the proper timing and efficiency of cleavage and processing of these sequences in ITS1 must depend upon the pre-rRNA substrates presenting the appropriate conformation to be recognized and acted upon by the nucleases. Curiously, five A3
factors, as well as r proteins L8 and L15, are associated with pre-rRNA near the 3′-end of 5.8S rRNA, perhaps some distance from ITS1 located upstream of the 5′-end of 5.8S rRNA (29
). It seems likely that these seven proteins, which bind RNA but lack any apparent enzymatic activities, may function in these processing steps by establishing conformations of pre-rRNA to create long-range interactions between the 5′-and 3′-ends of 5.8S rRNA, analogous to allosteric interactions that occur within the ribosome during protein synthesis (36
). As pointed out by Granneman et al.
), the A3
factors may coordinate folding and processing at the 5′-and 3′-ends of 5.8S rRNA to ensure that ITS1 processing occurs after proper folding of ITS2, and before removal of ITS2.
We recently found that Ebp2 and Rrs1 are localized at the nuclear periphery as well as the nucleolus and play roles in telomere organization and silencing (37
). Although it remains to be elucidated if Brx1 has similar functions, the synthetic lethality of brx1
reflects cooperative functions of the two proteins in ribosome biogenesis, not in other functions, because suppression of a defect in telomere organization of ebp2-14
did not suppress either a defect in temperature sensitivity for growth or a defect in ribosome biogenesis (37