Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

Abstract

Abstract. We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM/, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM/mRNA. The consequences of overexpressing NOP2 from the GAL/0 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure. T HE nucleolus is the specialized region within the nucleus where the majority of the steps in the complex process of ribosome subunit synthesis are executed (for recent yeast reviews, see Raue and Planta, 1991;Woolford and Warner, 1991). Within the nucleolus RNA polymerase I synthesizes a precursor rRNA, which is processed and modified, but not spliced, in a series of steps to generate mature 5.8 S, 18 S, and 25 S rRNAs. The 5 S rRNA is transcribed from a separate transcription unit by RNA polymerase III. The large subunit is assembled from probably up to 45 different ribosomal proteins and the 5 S, 5.8 S, and 25 S rRNAs, whereas the small subunit contains 32 ribosomal proteins and the 18 S rRNA. The biogenesis of ribosomal subunits in the nucleolus is thought to involve the coordinated formation of a series of subunit precursors consisting

Ytm1, Nop7, and Erb1 Form a Complex Necessary for Maturation of Yeast 66S Preribosomes

“…(A) Oligonucleotide probes or primers used to detect rRNAs and pre-rRNAs. (B) Yeast strains JWY3400 (YTM1) and JWY7128 (ytm1-1) were grown in YEPD medium at 25°C and shifted to 37°C for 5 h. Cells were pulse-labeled with [5,6 3 H]uracil for 5 min and chased with an excess of unlabeled uracil for 2, 5, 10, and 60 min. Equal cpm of RNA isolated from cells at each time point were subjected to electrophoresis on agarose-formaldehyde gels to separate each pre-rRNA or rRNA and detected by autoradiography.…”

Nop2p Is Required for Pre-rRNA Processing and 60S Ribosome Subunit Synthesis in Yeast

“…Cultures were grown at 30ЊC and diluted with prewarmed medium when OD 600 values reached 0.6 to 0.8. Total cellular protein was extracted in the presence of trichloroacetic acid, precipitated, separated on a 10.5% polyacrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by immunoblotting with affinity-purified polyclonal antibody APpAb3 against Nop2p (10). Monoclonal antibody 1G1 (2), which is specific for Pab1p, was used subsequently on the same blot.…”

“…A ClaI-BglII nop2::LEU2 disruption fragment from pBH45 was used to transform YBH12. Two 5-FOA-sensitive isolates (YBH3 and YBH4) obtained from Leu ϩ Ura ϩ transformants were analyzed by Southern blotting as previously described (10). To introduce plasmid-borne GAL-NOP2 into this background, pBH46 and pBH47 were constructed (Table 2).…”

“…Nop2p is a yeast nucleolar protein that is specifically upregulated during the onset of growth and is homologous to nucleolar protein p120 (10). p120 is a human proliferation marker that is predicted to function as a methylase (14,25).…”

GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.

“…The se-+ quence of ORF2 is identical to that of the NOP2 + gene, identified recently in a sequence deposited in GenBank (accession no. X82656) by de Beus et al (1994). The top line depicts the genomic DNA insert in pMG1; below that are depicted fragments present in subclones constructed from pMG1.…”