Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
J Biol Chem. 2009 Dec 11;284(50):35079-91. doi: 10.1074/jbc.M109.040774. Epub 2009 Sep 29.
Many RNA nucleases and helicases participate in ribosome biogenesis, but how they cooperate with each other is largely unknown. Here we report that in vivo cleavage of the yeast pre-rRNA at site D, the 3'-end of the 18 S rRNA, requires functional interactions between PIN (PilT N terminus) domain protein Nob1 and the DEAH box RNA helicase Prp43. Nob1 showed specific cleavage on a D-site substrate analogue in vitro, which was abolished by mutations in the Nob1 PIN domain or the RNA substrate. Genetic analyses linked Nob1 to the late pre-40 S-associated factor Ltv1, the RNA helicase Prp43, and its cofactor Pfa1. In strains lacking Ltv1, mutation of Prp43 or Pfa1 led to a striking accumulation of 20 S pre-rRNA in the cytoplasm due to inhibition of site D cleavage. This phenotype was suppressed by increased dosage of wild-type Nob1 but not by Nob1 variants mutated in the catalytic site. In ltv1/pfa1 mutants the 20 S pre-rRNA was susceptible to 3' to 5' degradation by the cytoplasmic exosome. This degraded into the 3' region of the 18 S rRNA, strongly indicating that the preribosomes are structurally defective.
许多 RNA 核酸酶和解旋酶参与核糖体生物发生，但它们彼此如何合作在很大程度上是未知的。在这里，我们报告说，酵母前 rRNA 在 D 位点的体内切割，即 18S rRNA 的 3'端，需要 PIN（PilT N 端）结构域蛋白 Nob1 和 DEAH 盒 RNA 解旋酶 Prp43 之间的功能相互作用。Nob1 在体外对 D 位点底物类似物表现出特异性切割，该切割被 Nob1 PIN 结构域或 RNA 底物中的突变所消除。遗传分析将 Nob1 与晚期前 40S 相关因子 Ltv1、RNA 解旋酶 Prp43 及其共因子 Pfa1 联系起来。在缺乏 Ltv1 的菌株中，Prp43 或 Pfa1 的突变导致由于抑制 D 位点切割而导致细胞质中 20S 前 rRNA 的惊人积累。该表型被增加的野生型 Nob1 的剂量所抑制，但不能被 Nob1 的催化位点突变的变体所抑制。在 ltv1/pfa1 突变体中，20S 前 rRNA 易受细胞质 exosome 的 3' 到 5' 降解。这降解成 18S rRNA 的 3' 区域，强烈表明前核糖体在结构上有缺陷。
