Daugeron M C, Linder P
Département de Biochimie Médicale, CMU, 1 Rue Michel Servet, CH-1211 Genève 4, Switzerland.
Nucleic Acids Res. 2001 Mar 1;29(5):1144-55. doi: 10.1093/nar/29.5.1144.
RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0). Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.
DEAD盒家族的RNA解旋酶几乎参与了所有涉及RNA分子的细胞过程。在此，我们描述了酵母RNA解旋酶Dbp8p（YHR169w）的功能特性。我们的结果表明，Dbp8p是小核糖体亚基生物合成所必需的一种核仁蛋白。体内Dbp8p的缺失导致核糖体亚基失衡，原因是40S核糖体亚基不足。随后通过脉冲追踪标记、Northern杂交和引物延伸对前体rRNA加工进行分析，结果显示，35S前体在A(1)和A(2)位点的早期切割步骤受到抑制，在A(0)位点延迟。因此，在没有Dbp8p的情况下，40S亚基的RNA部分18S rRNA的合成被阻断。通过对该蛋白家族具有酶活性的一些保守基序进行定点诱变，导致Dbp8p体内功能丧失，这有力地支持了Dbp8p作为一种真正的RNA解旋酶参与核糖体生物合成的观点。
