Ribosome biogenesis in eukaryotes involves a large number of factors, many of them identified during recent large-scale co-purification studies (16–18
). While these results were crucial to define the number of different proteins involved in this conserved pathway, they are not suitable for a detailed characterisation of the different intermediate ribosome precursors. Novel approaches are needed to address this question and one of them is the study of changes in the protein composition of the intermediate particles under mutant conditions (2
). We show here that coupling TAP purifications with carefully chosen mutants and novel methods of quantitative mass-spectrometry like SILAC (12
) allows the description of groups of pre-ribosomal factors that are likely to be functionally related. The definition of such groups may lead to oriented experiments that would help the comprehension of the involved molecular mechanisms.
We integrated our results about the assembly of pre-ribosomal factors on pre-60S particles in a simple schematic (A). Several groups of pre-60S factors could be defined, on the basis of the three analysed mutant effects. The earliest group of pre-60S factors is defined by the proteins that accumulate during Ebp2 depletion. Since this mutant accumulates 27SA2 pre-rRNA, the first form of RNA that is specific to the 60S assembly pathway and signals the separation from pre-40S particles, proteins of this group are likely to be already present on the earliest nascent 60S precursors.
Figure 7. SILAC-based definition of pre-60S sub-complexes. (A) Based on the results of our analysis we deduced possible association and dissociation steps that define several groups of pre-60S factors, relative to the involvement of Ebp2, Nog1/Nsa2 and Nog2 in (more ...)
One of the most interesting findings of this study was that some pre-60S factors, like Nop16, decreased in the purified complexes when Ebp2 was depleted while they accumulated under Nog1 or Nsa2 depletion, whatever the protein used for the precursors purification. The simplest explanation for this behaviour is that proteins from this intermediate group associate to the 60S precursors at a step that precedes the one blocked by the absence of Nog1 or Nsa2, and follows the one blocked by Ebp2 absence (A). The proteins that, like Mrt4, associate to the nuclear pre-60S particles at late steps depending on the action of Nog1 and Nsa2 can be further classified in two sub-groups, including in the latest one the factors that also depend on Nog2 for association to the pre-60S particles (for instance, Rsa4).
Our data cannot indicate with precision the moment of dissociation for most of the identified proteins because the lack of identification of a protein in a given complex is not an absolute proof for its absence from that complex. However, when a protein could be identified under some conditions in a complex but was absent from similar complexes purified with another bait protein, such information was pragmatically used to differentiate subgroups of pre-60S factors. With the above limitations in mind, we distinguished the proteins that looked specific to the early Mak11-associated complexes (for instance Dbp7) from those that were also present on later Rlp24 or Nog1-associated pre-60S particles (for instance Mak5). Except for known shuttling factors that are exported to the cytoplasm, the estimation of the point where other factors leave the particles would need further validation by specific experiments.
Our analysis provides new insights into the assembly scenario for the eukaryotic large ribosomal subunit in yeast, by describing the protein composition of distinct assembly intermediates along the pathway. The results were in excellent correlation with the topology of the network of physical association of pre-60S factors. Proteins that were enriched or decreased in precursor particles under mutant conditions were clustered in specific regions of the graph, depending on the analysed protein depletion (B, C and D). Our results are also in agreement with previously published data about the pre-60S factors function or association to the pre-ribosomal complexes. For instance, Dbp7, Dbp9, Nop4, Noc2, Rpf2, Ebp2, Rrp1 and Ssf1 were known for their participation in early steps of the large ribosomal subunit biogenesis (8
), and Rrp5 is a component of the SSU-processome (1
). Nug1 was characterised for its functions in the late steps of biogenesis, at the same time as Nog2 (28
) and Arx1 are both involved in the export of pre-60S particles from the nucleus (34
On the basis of the analysis of three distinct 60S biogenesis steps (depending on Ebp2, Nog1/Nsa2 and Nog2) we were thus able to obtain an unbiased panorama of the association timing for 30 pre-60S factors during ribosome biogenesis. These results, integrated into the physical association map for all the previously identified pre-60S proteins, and combined with data about the molecular function of each pre-60S factor in the biogenesis, are useful to build a view of protein dynamics in the ribosome biogenesis pathway.