Wu P, Brockenbrough J S, Metcalfe A C, Chen S, Aris J P
Department of Anatomy and Cell Biology, Health Science Center, Box 100235, University of Florida, Gainesville, Florida 32610, USA.
J Biol Chem. 1998 Jun 26;273(26):16453-63. doi: 10.1074/jbc.273.26.16453.
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 degreesC. 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 A0 and A2 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 co-immunoprecipitated 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.
我们鉴定出一种新型核仁蛋白Nop5p,它对酿酒酵母的生长至关重要。单克隆抗体B47和37C12可识别Nop5p,其预测大小为57 kDa,在其羧基末端具有KKX重复基序。去除KKX基序的截短形式具有功能且定位于核仁,但在37℃时生长缓慢。Nop5p与酵母Sik1p/Nop56p以及古细菌、植物和人类中的推定同源物具有显著的序列同源性。在GAL-NOP5菌株中耗尽Nop5p会使倍增时间延长约5倍,并相对于游离60 S亚基和25 S rRNA的水平选择性降低40 S核糖体亚基和18 S rRNA的稳态水平。Northern印迹和引物延伸分析表明,Nop5p的耗尽会损害35 S前体rRNA在A0和A2切割位点的加工。Nop5p与小核仁RNA U3、snR13、U14和U18相关。Nop5p的耗尽导致核仁蛋白Nop1p(酵母纤维蛋白原)定位于细胞核和细胞质中。此外,37C12可共免疫沉淀Nop1p。这些结果表明,Nop5p与Nop1p共同作用于早期前体rRNA加工步骤,这些步骤导致18 S rRNA的形成。