We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37°C. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A 0 and A 2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 coimmunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.Most of the steps of ribosome biogenesis in eukaryotic cells take place in the nucleolus. In the yeast Saccharomyces cerevisiae, a single long 35 S pre-rRNA is transcribed by RNA polymerase I and processed to 18 S, 5.8 S, and 25 S rRNAs through a series of co-and post-transcriptional steps. Ribosomal proteins imported from the cytoplasm are assembled with pre-rRNAs to form the small 40 S subunit and the large 60 S subunit. The 5 S rRNA is transcribed by RNA polymerase III from a separate transcription unit and is incorporated into the large subunit along with the 5.8 S and 25 S rRNAs, while 18 S rRNA is incorporated into the small subunit. During transcription and processing of pre-rRNA, a number of nucleotides are modified, primarily by the addition of 2Ј-O-methyl groups or by the formation of pseudouridine residues. The processing and modification of pre-rRNAs require non-ribosomal nucleolar proteins, many of which are associated with small nucleolar RNAs (snoRNAs) 1 in the form of small nucleolar ribonucleoprotein (snoRNP) complexes (reviewed in Refs. 1 and 2).The earliest processing events are those involved in the removal of the promoter proximal 5Ј-externally transcribed spacer (5Ј-ETS). Cleavage occurs at two sites within the 5Ј-ETS: at A 0 , in the middle region of the 5Ј-ETS; and at A 1 , which results in the formation of the 5Ј-end of the mature 18 S rRNA (reviewed in Ref. 3). Formation of 18 S requires processing to form its 3Ј-end, which involves processing at site A 2 in the first internally transcribed spacer (ITS1) followed by processing at site D, which yields the 3Ј-end (see Fig. 9). In yeast, many gene products are required for, or participate in, cleavage at sites A 0 , A 1 , and A 2 , attesting to the complex nature of this process. The yeast RNase III encod...

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