A molecular genetic approach using the yeast S. cerevisiae
was previously used in this laboratory (29
) to investigate whether eIF6, originally isolated and characterized on the basis of its in vitro ability to bind to 60S ribosomal subunits (17
) and prevent its association with the 40S ribosomal subunits, indeed functions as a translation initiation factor in vivo in yeast cells. Using yeast strain KSY603, which contains a conditional eIF6 expression system, we were able to remove eIF6 rapidly from yeast cells and to measure various parameters of protein synthesis in vivo and in vitro (29
). We observed that removal of eIF6 from yeast cells caused a progressive inhibition in the rate of protein synthesis in yeast cells. However, this inhibition of protein synthesis was due not to a defect in translation initiation but rather to selective reduction of the level of 60S ribosomal subunits in yeast cells, causing a stoichiometric imbalance between 40S and 60S ribosomal subunits with consequent formation of half-mer polysomes (29
). Similar observations were reported by Sanvito et al. (26
), who identified eIF6 from mammalian cells as a β4 integrin-interacting protein (3
). The question, however, remained: how does eIF6 function to maintain the steady-state level of 60S ribosomal subunits in yeast cells?
Experiments presented here show that the stability of mature 60S particles synthesized in KSY603 cells in the presence of eIF6 was not significantly affected following removal of eIF6 from these cells. Rather, our evidence clearly indicates that in eIF6-depleted cells, the reduction of 60S subunit levels is due to a primary defect in pre-rRNA processing. A pulse-label shortly after eIF6 depletion led to relatively normal incorporation into rRNA molecules and the expected, if slightly delayed, processing to 27S and 20S pre-rRNA species. However, while the 20S precursor RNA was processed to 18S rRNA efficiently, most of the 27S pre-rRNA species was degraded without forming 25S and 5.8S mature rRNAs.
These results were confirmed by Northern blot analysis. In eIF6-depleted cells, there is an increase in the steady-state level of 35S and 27SB precursor rRNAs and a marked decrease in the level of mature 25S and 5.8S rRNAs as well as of 7S pre-rRNA. This result reflects a defect in the processing of 27SB species to mature 25S rRNA and 7S pre-rRNA. Whereas depletion of eIF6 leads to a marked reduction in the steady-state level of 25S rRNA, presumably due to dilution, there was only a slight decrease in the amount of 18S rRNA during this time period. This observation points to a rather specific and immediate block in the synthesis of 60S-specific rRNAs in eIF6-depleted cells. Taken together, these results suggest that eIF6 is necessary for the formation of 60S subunits because it is necessary in the formation of 25S and 5.8S rRNAs. Perhaps the absence of eIF6 induces the processing machinery to make an error that results in immediate degradation of the 27S RNA. Alternatively, eIF6 may be needed to signal to the degradation machinery that the 60S particle is functional.
We do not know whether accumulation of 35S and 23S pre-rRNAs in eIF6-depleted cells results directly from eIF6 depletion or is a consequence of the defective 27SB pre-rRNA processing which causes some form of feedback inhibition or delayed processing of 35S pre-rRNA. Inhibition of processing of the 35S pre-rRNA, accumulation of the aberrant 23S pre-rRNA by cleavage of 35S RNA at site A3
, and reduced steady-state levels of 20S pre-rRNA have all been reported for many mutants that affect biogenesis of 60S ribosomal subunits (2
; see also reference 35
). These observations suggest that delayed processing at sites A0
may be a general feature of mutations that inhibit the formation of mature 25S and 5.8S rRNAs. It should also be noted that the pre-rRNA processing defects observed in eIF6-depleted cells closely resemble those previously reported for yeast cells lacking Nip7p, Nop2p, and Dbp3p (10
). Depletion of each of Nip7p and Nop2p in yeast cells caused a block in the processing of 27SB pre-rRNA to 25S rRNA and 7S pre-rRNA, while cells lacking Dbp3p were shown to be defective in the processing of 27SA2
pre-rRNA, resulting in the inhibition of synthesis of mature 25S and 5.8S rRNAs; these cells also accumulated 35S and 23S pre-rRNAs and reduced levels of 20S pre-rRNA. Reduction in the level of 20S pre-rRNA, in turn, causes some inhibition in the formation of 18S rRNA. It has been postulated (35
) that the requirement for 25S and 5.8S processing proteins for 18S rRNA synthesis is indirect. It is likely that the pre-rRNA processing machinery is a single large complex formed well before the specific steps in 25S and 5.8S rRNA formation. Thus, defects in any processing protein might inhibit early steps including eventually 18S rRNA synthesis.
It is now well established that the synthesis of ribosomal proteins and assembly of ribosomes are tightly coupled to processing and modification of pre-rRNA. Mutation or depletion of proteins involved in ribosome biogenesis usually leads to defects at multiple steps in the pathway of synthesis of ribosomes (12
). Thus, our experiments do not rule out the possibility that eIF6, in addition to being required for efficient processing of pre-rRNA, is required for the synthesis of ribosomal proteins and/or their assembly into mature ribosomes. It should be noted that measurement of the rate of transcription of the rRNA genes using the nuclear run-on assays described by Elion and Warner (8
) did not show any significant difference in the synthesis of 35S rRNA between the wild-type and eIF6-depleted cell extracts (data not shown).
In this work, we have also studied the subcellular localization of eIF6 in yeast cells. Although eIF6 is associated with free cytoplasmic 60S subunits both in mammalian cells (17
) and in the yeast S. cerevisiae
), indirect immunofluorescence studies using eIF6-HA fusion protein as well as cell fractionation studies clearly show that the protein is localized both in the cytoplasm and in the nucleus. These localization data are consistent with the role of eIF6 in pre-rRNA processing, mainly a nucleolar event. However, the predominant cytoplasmic localization of eIF6 suggests that eIF6 may have a function in the cytoplasm. It is tempting to speculate that eIF6 is exported from the nucleus in association with 60S ribosomal subunits and is released from the 60S subunit before or after it binds to the 40S initiation complex to form the 80S initiation complex. This is in accord with our observation that HA-tagged eIF6 expressed in yeast cells associates with free cytosolic 60S subunits and is not part of 80S monosomes or polyribosomes (29
). Association of eIF6 with free cytoplasmic 60S subunits suggests that eIF6 may act as a chaperone in transporting 60S subunits from the nucleus to the cytoplasm. The possibility also exists that although eIF6 does not directly function as a general translation initiation factor, its binding to 60S ribosomal subunits may serve as a checkpoint of the subunit joining step during initiation of protein synthesis. We found some time ago (34
) that 60S ribosomal subunits containing bound mammalian eIF6 are incapable of joining the 40S initiation complex to form the 80S initiation complex. These observations suggest that a mechanism must exist for the release of eIF6 from the 60S subunit either prior to or concomitant with the joining of the 60S subunit to the 40S initiation complex. Interestingly, Nip7p (39
) and Nmd3p (9
) have also been identified as proteins required for 60S biogenesis that also associate specifically with free 60S subunits in the cytoplasm of yeast cells. It remains to be seen whether these proteins possess a ribosomal subunit antiassociation activity similar to that observed for eIF6 (29
). On the other hand, the cytoplasmic localization and association of eIF6 with 60S ribosomal subunits might have no functional consequence or might be involved in a yet unknown function of eIF6.
eIF6 is an evolutionarily conserved protein. Proteins structurally homologous to eIF6 have also been found in phylogenetically distant species including archeaons and plants but not in eubacteria (3
). The high degree of conservation in amino acid sequence among phylogenetically distant species indicates that eIF6 plays an important role in an essential process in the cell.