Ho J H, Johnson A W
Department of Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
Mol Cell Biol. 1999 Mar;19(3):2389-99. doi: 10.1128/MCB.19.3.2389.
A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.
发现在缺乏XRN1的情况下，NMD3中的突变是致死性的，XRN1编码负责mRNA周转的主要细胞质外切核糖核酸酶。对NMD3的分子遗传学分析表明，它是稳定的60S核糖体亚基所需的必需基因。携带NMD3温度敏感等位基因的细胞在非允许温度下60S亚基水平降低，这导致了半聚体多核糖体的形成。rRNA生物合成的脉冲追踪分析表明，25S rRNA的合成和加工动力学与野生型动力学相似。然而，成熟RNA迅速降解，半衰期为4分钟。Nmd3p作为一种细胞质蛋白进行分级分离，并在蔗糖梯度中沉降在游离60S亚基的位置。这些结果表明，Nmd3p是60S亚基细胞质后期组装步骤所需的细胞质因子，但不是核糖体蛋白。Nmd3p的假定直系同源物存在于果蝇、线虫和古细菌中，但不存在于真细菌中。Nmd3蛋白序列不包含易于识别的已知功能基序。然而，这些蛋白质都有一个氨基末端结构域，包含四个Cx2C重复序列，让人联想到锌结合蛋白，与核酸结合或蛋白质寡聚化有关。
