Colley A, Beggs J D, Tollervey D, Lafontaine D L
Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, Scotland.
Mol Cell Biol. 2000 Oct;20(19):7238-46. doi: 10.1128/MCB.20.19.7238-7246.2000.
Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A(0), A(1), and A(2), while Dhr1p depletion inhibited cleavage at sites A(1) and A(2). No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5' end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.
推定的RNA解旋酶参与基因表达的多个方面。推定的RNA解旋酶DEAH-box家族中所有先前已表征的成员都参与前体mRNA剪接。在此，我们报告了对两种新型DEAH-box RNA解旋酶Dhr1p和Dhr2p的分析，发现它们主要定位于核仁。这两个基因对于细胞存活至关重要，因此构建了受MET调控的等位基因。在体内或体外，Dhr1p或Dhr2p的缺失对前体mRNA剪接均未产生可检测到的影响。然而，18S rRNA的合成需要Dhr1p和Dhr2p两者。Dhr2p的缺失抑制了前体rRNA在A(0)、A(1)和A(2)位点的切割，而Dhr1p的缺失则抑制了A(1)和A(2)位点的切割。未检测到ProtA-Dhr2p与snoRNAs的共沉淀，但Dhr1p-ProtA与U3 snoRNA稳定结合。Dhr1p的缺失抑制了需要U3与18S rRNA 5'端碱基配对的加工步骤。我们推测，Dhr1p通过U3-18S rRNA相互作用被靶向至前核糖体颗粒，并且在中央假结形成过程中rRNA的结构重组需要该酶。
