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Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A 0 through A 2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5 external transcribed spacer (ETS) and the box C؉D snoRNA U3, as well as additional box C؉D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5 ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.Ribosome biogenesis is a complicated pathway that requires in eukaryotes an excess of 200 protein trans-acting factors and just as many small nucleolar RNAs (snoRNAs) (27,28,30,38). The process is initiated and largely takes place in the nucleolus, a dedicated subcellular compartment. RNA polymerase I (Pol I) transcription generates a large precursor (35S in Saccharomyces cerevisiae) encoding three out of the four mature rRNAs (5.8S, 18S, and 25S rRNAs). Mature sequences are released from noncoding spacer regions (5Ј and 3Ј ETS and ITS1 and ITS2), following a relatively well-established processing pathway involving both endoribonucleolytic and exoribonucleolytic digestions (Fig. 1A) (reviewed in references 30 and 45). 5S rRNA is encoded by a Pol III transcript and matured independently.Most ribosome biogenesis factors are localized to the nucleolus, are essential for cell viability, and are components of large preribosomal protein complexes. Consequently, proteomic analyses have resulted in the identification of many potential new rRNA processing factors (RRPs) (reviewed in references 10 and 19), and the use of affinity purification has delineated distinct and presumably successive preribosomal complexes (reviewed in references 10, 12, and 46). Among these is the small-subunit (SSU) processome (3, 4, 9, 14, 35), a U3-containing ribonucleoprotein complex primarily involved in the three initial pre-rRNA processing reactions (cleavage at sites A 0 through A 2 ) (see Fig. 1A) and therefore required for 18S rRNA synthesis.As 35S pre-rRNA molecules emerge from the Pol I transcription machinery, 5Ј pre-rRNA termini are bound by U3 and early RRPs, generating the so-called "terminal knobs....

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