In this work, we have addressed the functional characterisation in ribosome biogenesis of P0, the central component of the r-stalk, and its homologous r-protein-like Mrt4. The architecture of this essential r-domain has been extensively studied mainly in prokaryotes by distinct techniques, which evaluate the interactions between its components (39
). The eukaryotic r-stalk structure is less stable than its prokaryotic counterpart and seems to modulate the cell translation activity through a mechanism that involves the exchange between ribosome-bound and free cytoplasmic stalk components (42
). Unfortunately, there is little information available on the role of P0 and its prokaryotic counterpart L10 in ribosome biogenesis. In eukaryotes, a notable sequence homology exists between P0 and the non-essential ribosome synthesis factor Mrt4, which suggests a functional relationship between both proteins (49
). In yeast, the absence of either P0 or Mrt4, which is lethal only in the first case, leads to a deficit of active 60S relative to 40S r-subunits [(10
) and ]. In agreement with this information, we show herein that both proteins are required for the production of 25S and 5.8S rRNAs ( and Figure S2
); deletion of MRT4
and depletion of P0 affects pre-rRNA processing leading to a discrete accumulation of 35S and upon early times of P0 depletion also of aberrant 23S pre-rRNA. This is a general feature in many strains affected in 60S r-subunit biogenesis (1
) that most likely occurs due to failure of recycling of trans
-acting factors required for the early pre-rRNA cleavages at sites A0
that improperly dissociate from defective pre-60S r-particles (13
). Most importantly, the absence of Mrt4 and the depletion of P0 lead to reduced steady-state levels of all 27S and 7S pre-rRNAs and, therefore, of mature 25S and 5.8S rRNAs. In general, the pre-rRNA processing defects detected upon depletion of P0 were more drastic than those after the deletion of MRT4
. In addition, depletion of P0 is epistatic over and synergistic with the absence of Mrt4, confirming the functional relationship between the two proteins. Since it is unlikely that either P0 or Mrt4 have a direct
role in 27S pre-rRNA processing reactions, we assume that the depletion of P0 and the absence of Mrt4 lead to defective assembly of early pre-60S r-particles, which causes destabilisation and efficient degradation of the 27S pre-rRNAs and their products. More relevant concerning the final ribosome yield of the conditional systems tested is the fact that P0 depleted but not MRT4
deleted cells accumulated Rpl25-eGFP within the nucleus (). Rpl25 has been suggested as assembling early during ribosome biogenesis (28
). This result suggests a block in nucleo-cytoplasmic export of defective pre-60S r-particles that lack P0 and/or other factor limited upon P0 depletion but not of pre-60S r-particles assembled in the absence of Mrt4.
Mrt4 has been previously shown to localize to the nucleolus (48
) and found to be associated with many pre-60S r-particles (10
). Moreover, Mrt4–TAP complexes co-enriched defined pre-60S r-particles (). Analysis of the RNA composition of these complexes indicates that Mrt4 might bind to pre-60S r-particles soon after formation of 27SB pre-rRNAs and apparently dissociate only when pre-rRNA processing reactions have been completed. At steady-state levels, Mrt4 appears to stably concentrate in medium/late pre-60S r-particles, which contains 7S pre-rRNA ( and ). The protein composition of the Mrt4–TAP complexes also reflects this fact and indeed most of the trans
-acting factors and r-proteins identified have been previously listed as components of Nug1–TAP complexes, which are considered the representatives of nuclear medium pre-60S r-particles (11
). On the other hand, our heterokaryon assay data strongly suggest that Mrt4 travels associated with those nuclear pre-60S r-particles that exit to the cytoplasm (). There, Mrt4 dissociates from the particles and is efficiently reimported to the nucleus in order to maintain its steady-state nuclear distribution. Shuttling factors often require other proteins to be released from the cytoplasmic pre-60S r-particles and/or recycled back to the nucleus (20
), amongst them, Drg1, which is an ATPase required for the release of Arx1, Rlp24, Nog1 and Tif6 (23
), and Efl1/Ria1 and Lsg1, which are GTPases required for optimal dissociation of Tif6 and Nmd3, respectively (22
). Little is known about Mrt4 releasing factors; our data suggest that the GTPase Nug1 does not seem to have this role since Mrt4 does not mislocalize upon loss of its function. Very recently, while this manuscript was being prepared, Johnson and co-workers have shown that the efficient release of Mrt4 from pre-60S r-particles requires the participation of the non-essential phosphatase Yvh1 that associates with late pre-60S r-particles (A.W. Johnson, personal communication).
When and how is P0 assembled? We have previously shown that P0 and Mrt4 interact in a mutually exclusive manner to the 25S rRNA GAR domain (49
). Taking this into account, a simple model where Mrt4 and P0 successively occupy the GAR domain in pre-60S r-particles and mature 60S r-subunits, respectively, can be envisaged for the timing of P0 assembly. Since our data strongly suggest that Mrt4 dissociates from late, most likely cytoplasmic, pre-60S r-particles, the exchange of Mrt4 with P0 might take place mainly in the cytoplasm, which is agreement with the interpretation of LMB experiments. Thus, if P0 predominantly assembled in the nucleus, we would expect the export of P0-containing pre-60S r-particles to be Crm1-dependent in the LMB-sensitive strain. However, functional GFP-tagged P0 did not relocalize to the nucleus in the presence of LMB even in the absence of Mrt4 (). Moreover, we show here that, in wild-type cells, P0 is practically absent from some Mrt4-containing medium pre-60S r-particles such as the Nug1–TAP complexes (). These results are in agreement with those previously reported for Nog1-, Nog2- and Rlp24–TAP complexes (62
), Nsa1-defined pre-60S r-particles (55
) and Nsa3–TAP and Ssf1–TAP complexes (11
), all of which lack P0 as tested using either mass-spectrometry or specific antibodies. On the contrary, P0 is clearly present in late complexes such as the ones obtained using Arx1–TAP as bait, which contain little Mrt4 and cytoplasmic complexes such as the Lsg1–TAP ones, which lack Mrt4 ().
Our data also demonstrate that release of Mrt4 from pre-60S particles requires the presence of P0. This conclusion is based on the fact that Mrt4-eGFP mislocalized to the cytoplasm upon P0 depletion (), where it is still associated with high-molecular-mass complexes, most likely inactive almost mature 60S r-subunits lacking P0 (B and C). These complexes strongly accumulated increasing the free 60S peak in polysome profiles even in the absence of bound Mrt4 (D and F). Our experimental approaches do not address whether Mrt4 is preferentially released from pre-60S r-particles via a direct displacement by P0 or via a trans-acting factor. As above mentioned, it has been recently shown that Yvh1 is required for removing Mrt4 from late pre-60S r-particles that are then susceptible to bind P0 in the cytoplasm (A.W. Johnson, personal communication).
In apparent contradiction with the previous data, which strongly support that an important part of P0 is assembled in the cytoplasm after an exchange with Mrt4, the Nop7–TAP purified complexes contain a significant amount of both Mrt4 and P0. The presence of both proteins in these particles reflect heterogeneous composition since it is known that Nop7–TAP complexes are a mixture of several early, medium and even late pre-60S intermediates (10
), see also (11
). Nevertheless, since Nop7 seems to be a nuclear protein, which is unable to shuttle between the nucleus and the cytoplasm (), some P0 must assemble in the nucleus. Accordingly, the study of the P0 assembly dynamics in the mrt4
null mutant also leads to interesting conclusions being the most obvious one that P0 does not strictly require Mrt4 for association with pre-60S r-particles. Strikingly, in mrt4
null cells, the amount of P0 increases in both Nop7-and Nug1–TAP particles to proportions close to that of mature ribosomes (), indicating that in the absence of Mrt4, the nuclear assembly of P0 is enhanced. Some of these particles might be subjected to surveillance and degradation, which provide an explanation for the net 60S r-subunit deficit in mrt4
null cells; however, since active ribosomes are still synthesized in these cells, this alternative Mrt4-independent P0 assembly pathway that may operate even in the presence of Mrt4 cannot be excluded.
Several arguments could help to understand the apparent discrepancy of the data supporting both a nuclear and a cytoplasmic assembly of P0: (i) the fraction of P0 that assemble in the nucleus could be minor versus that assembling in the cytoplasm and thus P0-eGFP should not be detected there after the LMB treatment, (ii) export of pre-60S r-particles containing P0 could be independent of Crm1, (iii) the GFP bait, although shown not to interfere with the function of P0 and its capability of association with nuclear Nop7–TAP complexes (C), could influence the import of the P0-eGFP protein in the nucleus after the treatment with LMB, as earlier discussed (21
). Further experiments are required to solve the questions raised; however, the possibility that ribosome biogenesis operates as a mesh of pathways rather than as a linear series of events has been previously suggested (12
). This is an appealing possibility as a source of eukaryotic ribosome heterogeneity (72
), which starts being considered on the bases of new ribosome-dependent translation regulatory mechanisms (42
). The existence of alternative assembly pathways, at least for some r-components, might also explain some of the paradoxical paralogue-specific effects recently reported for duplicated genes encoding r-proteins (74
). Figure S6
summarizes the conclusions of this study concerning the role of Mrt4 and the assembly of P0 in wild-type conditions, in the absence of Mrt4 or upon depletion of P0.
In Escherichia coli
, L10 is a component of the RI50[1
] complex, which is the first in vitro
reconstitution intermediate of 50S r-subunits (75
). In vivo
, it seems that L10 assembles in the p1
50S complex, which is the earliest of the three pre-50S r-particles detected (76
). Thus, the prokaryotic and eukaryotic scenarios seem to be different and it prompted us to speculate whether Mrt4 appeared during evolution to optimize ribosome assembly by preventing early
P0 assembly. Our structural models indicate that P0 binds a little tighter than Mrt4 to the GAR domain (49
); therefore, it is possible that the premature binding of P0 to early nuclear pre-ribosomal particles could impede its appropriate maturation by interfering in defined structural rearrangements within these particles. Additionally, replacement of an r-protein-like factor by its r-protein counterpart might provide directionality to the ribosome synthesis process throughout different subcellular regions (nucleolus, nucleoplasm, cytoplasm) and opportunities for quality control mechanisms. Mrt4-P0 is not the sole example of a paralogue pair comprised of a non-ribosomal factor and a r-protein. Factors Imp3, Rlp7, Rlp24 show significant homology to r-proteins Rps9, Rpl7 and Rpl24, respectively (24
). It would be appealing to understand whether or not all these pairs have arisen during evolution to deal with similar situations during ribosome biogenesis.