We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose-repressible promoter. After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate. This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2. Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed. These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.In all eukaryotes, rRNA is transcribed as a single long transcript and processed by cleavages, nucleotide modification, and exonucleolytic degradation to generate the mature rRNAs. In the yeast Saccharomyces cerevisiae, these reactions result in the production of the mature 18S, 5.8S, and 25S rRNAs, which are assembled with the 5S ribonucleoprotein (RNP) and ribosomal proteins to form mature ribosomes. These events take place in the cell nucleolus. A number of small nucleolar ribonucleoproteins (snoRNPs) are required for many of these processing steps (25,27,40,45).Because it was readily identified in both vertebrate and yeast cells, the U3 snoRNP has been studied in a number of different organisms. Functional studies on the role of the U3 snoRNP in pre-rRNA processing have been carried out with cell extracts from mouse cells and Xenopus oocytes and in vivo in Xenopus laevis oocytes and in S. cerevisiae (4,6,18,22,30,34). Collectively, the results from these experiments point to an obligate role for the U3 snoRNP in the cleavages in the 5Ј external transcribed spacer (ETS) and in internal transcribed spacer 1 (ITS1) that generate the 18S rRNA (A0, A1, and A2 in Fig. 6).The 5Ј-most U3-dependent cleavage site (A0) in yeast also requires the presence of the RNase III homolog, the Rnt1 protein (10). In fact, in vitro yeast RNase III will cleave at this site in the absence of any other factors. This suggests that RNase III is catalytic for this processing step and th...