Zanchin N I, Goldfarb D S
Department of Biology, University of Rochester, Rochester, New York 14627, USA.
Mol Cell Biol. 1999 Feb;19(2):1518-25. doi: 10.1128/MCB.19.2.1518.
NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001-5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3'-to-5' exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.
NIP7编码一种保守的酿酒酵母核仁蛋白，该蛋白是60S亚基生物合成所必需的（N. I. T. 赞钦、P. 罗伯茨、A. 德席尔瓦、F. 谢尔曼和D. S. 戈德法布，《分子与细胞生物学》17:5001 - 5015, 1997）。在双杂交筛选中，Rrp43p和另一种必需蛋白Nop8p被鉴定为与Nip7p相互作用的蛋白。通过在免疫球蛋白G - 琼脂糖上对Nip7p与蛋白A标记的Rrp43p和Nop8p进行共纯化，提供了相互作用的生化证据。缺乏Nop8p的细胞中游离60S核糖体和多核糖体水平降低，并积累了半聚核糖体。缺乏Nop8p的细胞还积累了35S前体rRNA和异常的23S前体rRNA。缺乏Nop8p的细胞未能积累25S或27S rRNA，尽管它们确实合成了大量的18S rRNA。这些结果表明，在去除包含18S rRNA的片段后，缺乏Nop8p的细胞中27S或25S rRNA会被降解。缺乏Nip7p的细胞表现出与缺乏Nop8p的细胞相同的缺陷，只是它们积累了27S前体。Rrp43p是外切体的一个组分，外切体是一种3'至5'核酸外切酶复合物，其亚基与5.8S rRNA加工和mRNA周转有关。绿色荧光蛋白（GFP） - Nop8p和GFP - Nip7p都定位于核仁，而GFP - Rrp43p定位于整个细胞核，在细胞质中的定位较少。不同池的Rrp43p可能既与外切体相互作用，也与Nip7p相互作用，可能在细胞核和细胞质中都有相互作用，以在60S核糖体生物合成和mRNA周转的多步骤过程中催化类似反应。
