Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis
Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis
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DOI：
10.1093/nar/29.5.1144
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发表时间：
2001-03-01
影响因子：
14.9
通讯作者：
Linder, P
中科院分区：
文献类型：
--
作者：
Daugeron, ML;Linder, P
DEAD盒家族的RNA解旋酶参与几乎所有涉及RNA分子的细胞过程。在这里，我们描述了酵母RNA解旋酶Dbp8p（YHR 169w）的功能特性。我们的研究结果表明，Dbp8p是一个必不可少的核仁蛋白所需的小核糖体亚基的生物合成。在体内耗尽Dbp8p导致核糖体亚基的不平衡，由于在40 S核糖体亚基的赤字。随后通过脉冲追踪标记、北方杂交和引物延伸对前体rRNA加工的分析表明，35 S前体在位点A（1）和A（2）的切割的早期步骤在位点A（0）被抑制和延迟。因此，在Dbp8p不存在的情况下，40S亚基的RNA部分18S rRNA的合成被阻断。Dbp8p作为一个真正的RNA解旋酶在核糖体生物合成中的参与得到了Dbp8p体内功能丧失的有力支持，这些功能是通过对携带蛋白质家族的酶特性的一些保守基序进行定点诱变获得的。
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.