Extracts were prepared from undepleted and Bfr2-depleted cells, and we carried out IPs followed by northern analyses (Figure 6). associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the 50S complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome. == INTRODUCTION == The making of eukaryotic ribosomes is an intricate process that is highly conserved. Our knowledge of ribosome biogenesis comes mainly from studies in the budding yeastSaccharomyces cerevisiae(14). Ribosome biogenesis initiates within the nucleolus, continues in the nucleoplasm and terminates in the cytoplasm. This process involves ribosomal RNA (rRNA) transcription, processing, modification and assembly of rRNAs with ribosomal proteins, which leads to the synthesis of the small and large ribosomal subunits (40S and 60S) (1,2,5,6). A key process in ribosome biogenesis is the production of mature rRNAs, the functional components of ribosomes (7). Yeast RNA polymerase I synthesizes a long precursor of 35S that encodes the 18S, 5.8S and 25S rRNAs, whereas the 5S rRNA is independently transcribed by RNA polymerase III (2,8). The 35S pre-rRNA is subjected to an orderly maturation process that requires about 200trans-acting factors (1,6,8,9). In addition, tens of small nucleolar RNAs (snoRNAs) base pair transiently with pre-rRNAs and direct site-specific post-transcriptional modification of rRNAs. Very few snoRNAs are required for the endonucleolytic cleavages that remove spacer sequences from pre-rRNAs. In yeast, only U3, U14 and snR30 snoRNAs are essential for the cleavage reactions that lead to the Lomitapide production of 18S rRNA (2,10,11). The functionally active U3 ribonucleoprotein (RNP) is a very large complex of 80S called thesmallsubunit (SSU) processome, which is formed at the 5 end of nascent pre-rRNA and can be seen under the electron microscope (12,13). In yeast, the SSU processome is implicated in early pre-rRNA cleavages at processing sites A0, A1 and A2 (12,13;Figure 1). The SSU processome is an early pre-ribosomal particle that is necessary for maturation of the 18S rRNA: it Lomitapide contains the U3 snoRNA and about 72 proteins including ribosome biogenesis factors and ribosomal proteins (14). These proteins assemble and interact together to form the SSU processome. A number of studies identified the presence of sub-complexes of the SSU processome. These sub-complexes are called UtpA/tUTP, UtpB, UtpC, Mpp10, Rcl1/Bms1 and U3 snoRNP (1525). However, proteins identified from these sub-complexes account for 43% of the proteins of the SSU processome, indicating that many proteins of the SSU processome have not yet been identified as components of a sub-complex (14,26). There are also studies showing that some of the sub-complexes of the SSU processome associate with the rRNA precursors in a hierarchical and stepwise manner (22,27,28). == Figure 1. == The pre-rRNA processing pathway in yeast. The structure of the 35S pre-rRNA (primary transcript) is shown on top. The rectangles represent cellular compartments in which different steps of the processing pathway take place. The pre-rRNA cleavage sites are indicated on the transcripts. Many of the non-ribosomal factors involved in rRNA maturation are RNA helicases. These enzymes are viewed as molecular motors that rearrange RNA structures in an energy-dependent fashion (2935). However, some can rearrange RNAprotein complexes, and many could in fact be RNPases (30,36). DEAD-box protein Dbp4 is a RNA helicase that is phylogenetically conserved and essential for yeast viability; Dbp4 was first identified as a multi-copy suppressor of lethal mutations in the Y domain of U14 snoRNA (37). More recently it was shown that Dbp4 is required for the production of 18S rRNA and more specifically for the early cleavages at sites A0, A1 and A2 of the pre-rRNA (38). The C-terminal extension that flanks the catalytic core of SERP2 Dbp4 contains a predicted coiled-coil motif, which is conserved in all Dbp4 orthologs (our unpublished observation). Because this motif is implicated in proteinprotein interactions, Dbp4 might function in a complex with other protein(s). We found that Dbp4 associates with the essential nucleolar proteins Bfr2 and Enp2 (3941). We also show that Bfr2 and Enp2 are implicated in the early cleavages leading to 18S rRNA production, and that Bfr2 and Enp2 associate with the U3 snoRNA and the U3-specific protein Lomitapide Mpp10. Sucrose gradient analyses and immunoprecipitation assays revealed that Dbp4, Bfr2 and Enp2 associate together in complexes of 50S and 80S. These proteins do not associate with the U3.