We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.Synthesis of the 40S and 60S ribosomal subunits in eukaryotes is a particularly intricate process that involves the synthesis of four ribosomal RNAs, their assembly with close to 80 ribosomal proteins, and transport of preribosomal particles from the nucleus to the cytoplasm where translation occurs (30,56,76,98). Eukaryotic ribosome synthesis requires the action of RNA polymerases I, II, and III that, respectively, transcribe a long common precursor to the 18S, 5.8S, and 25S rRNAs, the pre-mRNAs of ribosomal proteins, and the precursor to 5S rRNA. The pre-rRNA transcribed by RNA polymerase I contains, in addition to the sequences retained in the mature cytoplasmic ribosomes, long spacer regions that will be removed by a complex series of endo-and exonucleolytic cleavage steps (for a schematic representation of the pre-rRNA processing steps, see Fig. 4C). Moreover, specific nucleotides of rRNAs will undergo posttranscriptional chemical modification. By far the two most common modifications are the conversion of uridines into pseudouridines and the methylation of the oxygen at the 2Ј position of ribose moieties. These modifications are carried out by box H/ACA and box C/D small nucleolar RNPs (snoRNPs), respectively (4,26,28,49,50,86). Although the precise functions of modified nucleotides in rRNAs are not known, they are believed to significantly contribute to ribosome function since they are present in the most highly conserved and functionally important regions of rRNAs (15). Indeed, M. Fournier and collaborators have recently shown that lack of a single pseudouridine within the peptidyl transferase center, due to alteration of the box H/ACA snoRNP that produces this pseudouridine, is correlated with a substantial reduction in translational activity (48). Likewise, Bonnerot and colleagues have shown that the absence of 2Ј-O-ribose methylation of U2918 within the peptidyl transferase center increases sensitivity to paro...