In eukaryotic cells ribosomes are assembled in the nucleolus and must be transported through the nuclear pore complex to the cytoplasm, where they function in translation. The first discrete intermediate preribosomal species, the 90S particle (40
), assembles around the 35S primary transcript in the nucleolus and contains integral ribosomal proteins, trans
-acting rRNA processing factors, and subunit assembly factors. Recent evidence suggests that the 90S particle assembles cotranscriptionally on the primary transcript, is devoted to 40S biogenesis, and is almost entirely lacking in 60S proteins and assembly factors (5
). Cleavage of the transcript into 20S and 27S species in the nucleolus, the precursors of 18S and 25S, respectively, may release the pre-40S complex. The subsequent processing and assembly of the 66S particle, the nucleolar precursor of the 60S subunit (44
), appears to be independent of 40S assembly (5
). Release of the large subunit from the nucleolus into the nucleoplasm likely coincides with the release of many of the 66S-associated biogenesis factors (9
), yielding a nucleoplasmic pre-60S subunit.
Several rRNA processing and protein assembly events may be associated with or required for release of the pre-60S subunit from the nucleolus into the nucleoplasm. For example, the cleavage of 27S pre-rRNA into 25S and 7S RNAs precedes release into the nucleoplasm (15
). In addition, the nucleolar 66S particle contains the two proteins Rlp7p and Rlp24p that are similar to mature subunit proteins Rpl7p and Rpl24p, respectively (6
). Thus, it has been suggested that the exchange of Rpl7p and Rpl24p for Rlp7p and Rlp24p proteins is necessary for 60S subunit release (6
). Once in the nucleoplasm, the pre-60S particle undergoes further processing. For example, in yeast the 3′ trimming of 7S to 5.8S is likely a nucleoplasmic event (15
) that requires the exosome, Rex1p, Rex2p, and Ngl2p (8
). Finally, export to the cytoplasm requires the addition of the export adapter Nmd3p (11, 20) that provides the leucine-rich nuclear export sequence recognized by Crm1 in a Ran-GTP-dependent fashion. Based on genetic and physical evidence, Nmd3p may be recruited to the subunit by Rpl10p (11
). The temporal sequence and cellular localization of these events is not well established, and it is possible that some of these events are not obligatorily linked with a particular compartment of the nucleus or with other events happening in parallel on the subunit.
The large and small ribosomal subunits are produced in equimolar amounts since they are derived from a common precursor. Nevertheless, they are independently matured and exported from the nucleus, with the large subunit requiring a longer transit time in the nucleoplasm (15
). Recent advances by several research groups have provided significant insight into the nuclear export pathway of the 60S subunit. Release of the pre-60S subunit from the nucleolus requires Noc1p/Mak21p (YDR060W), Noc2p (YOR206W), and Noc3p (YLR002C) (28
). Although the functions of these proteins are not known, they appear to act in a sequential fashion, with Noc1p/Mak21p and Noc2p present in the nucleolus and Noc2p and Noc3p found in the nucleoplasm.
We and others have recently shown that the nuclear export signal (NES) for the large subunit is provided by the adapter protein Nmd3p (Yhr170wp). Expression of mutant Nmd3 protein lacking the NES inhibits export of the 60S subunit (11
). Genetic and physical interactions between Nmd3p and Rpl10p suggest that Rpl10p may constitute at least part of the binding site for Nmd3p on the 60S subunit (11
). The leucine-rich NES of Nmd3p is recognized by the karyopherin Crm1p (YGR218W) (11
). Consequently, export of 60S subunits is blocked in crm1
) conditional mutants (38
) or by the Crm1p inhibitor leptomycin B (LMB) (25
) in LMB-sensitive yeast (11
). This pathway for export of 60S subunits is conserved in metazoan cells (40a).
Nascent 60S subunits are incorporated into polysomes more slowly than are nascent 40S subunits. This likely reflects the slower processing of 60S subunits in the nucleus (39
), as well as a delay in the recruitment of 60S subunits into polysomes in the cytoplasm (45
). The reason for this cytoplasmic delay is not clear, but it could be due to the slow addition or removal of proteins from the nascent subunit, a conformational change of the subunit, or covalent modification of the rRNA and/or r-proteins.
To identify additional proteins that bind to the free 60S subunit and that act in the biogenesis and transport of the 60S subunit, we have purified Nmd3p-bound subunits. Among the nonribosomal proteins associated with these subunits were two monomeric GTP-binding proteins, Nog1p and Lsg1p. These two proteins are both essential and required for 60S biogenesis. We show that Lsg1p is a cytoplasmic protein, whereas Nog1p has recently been described as a nucleolar protein (33