The synthesis of ribosomes is one of the major metabolic pathways of a cell. In Saccharomyces cerevisiae
, ribosome assembly begins in the nucleolus after the transcription of two rRNA precursors, the 35S RNA (precursor of the 18S, 5.8S, and 25S rRNAs) and the pre-5S RNA, by RNA polymerases I and III, respectively. The synthesized pre-rRNAs are modified extensively at multiple positions specified by small nucleolar ribonucleoparticles (snoRNPs) or specific enzymes (1
). During rRNA maturation, the 5′ and 3′ external transcribed sequences (ETS) and internal transcribed sequence 1 (ITS1) and ITS2 are removed from the 35S precursor RNA by well-ordered cleavages and trimming events, which require the enzymatic activities of helicases and endo- and exonucleases (19
Cotranscriptional assembly of ribosomal and nonribosomal proteins in the nucleolus gives rise to a large ribonucleoprotein particle corresponding to the 90S preribosomal complexes described more than 20 years ago (35
) and recently characterized biochemically (8
). These early preribosomal complexes are further converted to smaller pre-40S (43S) and pre-60S (66S) particles, precursors of the mature small and large ribosomal subunits. The pre-40S complexes, each containing a precursor of the 18S rRNA, are exported into the cytoplasm, where they give rise to the mature 40S ribosomal particles (36
). Most of the large ribosomal subunit proteins are absent from the 90S preribosomes (8
) and associate in the nucleolus with the pre-rRNA, probably concomitantly with the formation of the pre-60S particles. During pre-60S particle maturation, 27S pre-rRNA intermediates are converted into 25S and 5.8S mature rRNAs by successive and well-ordered steps. Several pre-60S particles, which differ in their RNA and protein compositions, are generated successively in the nucleolus and nucleoplasm and then exported to the cytoplasm (for reviews see references 11
, and 37
The existence of ribosome precursors in the form of discrete particles during eukaryotic ribosome biogenesis was described many years ago in experiments using HeLa and yeast cells (for a review on these early results see reference 38
). However, a detailed characterization of the preribosomal complexes emerged only recently due to the development of affinity purification methods and large-scale protein identification by mass spectrometry (3
). In addition to these results, the high-throughput identification of protein complexes in yeast (13
) generated a large amount of data and offered a global view of the factors physically associated with preribosomal particles. To date, more than 120 nonribosomal proteins are predicted to be physically associated in large ribonucleoprotein complexes along the maturation pathway from the nucleolus to the cytoplasm. The physical association with the preribosomes is, for many of these conserved factors, the only hint as to their function.
Previous studies on preribosomal particle composition showed that different tagged proteins were associated with different sets of preribosomal factors in multiple preribosomal complexes (24
; for a review, see reference 11
). However, especially for early pre-60S particles of highly complex composition, little is known about the identities of the different particles and the association or dissociation of specific factors. A major limitation in the study of ribosome assembly comes from the difficulties in isolating homogenous preribosomal intermediates. By blocking ribosome biogenesis at specific steps and by analyzing the changes in the composition of early pre-60S intermediates, we were able to show that specific protein association and dissociation steps are required for the maturation of 27S-containing pre-60S particles during ribosome biogenesis. This novel approach should be able to elucidate the order of the assembly of preribosomal factors on, and their disassembly from, the particles and the mechanisms governing this coordinated process. We show here that direct protein-protein interactions are likely to be needed for the assembly of multiple complexes during ribosome assembly.