Sardana Richa, Zhu Jieyi, Gill Michael, Johnson Arlen W
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA
Mol Cell Biol. 2014 Jun;34(12):2208-20. doi: 10.1128/MCB.01656-13. Epub 2014 Apr 7.
The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23Δ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23Δ. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ∼45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function.
小核糖体亚基在新生的初级转录本上共转录组装。在A2位点的切割释放出前40S亚基。我们之前鉴定出Bud23是一种保守的真核甲基转移酶，它是在A2位点进行有效切割所必需的。在此，我们报告Bud23在物理和功能上与DEAH框RNA解旋酶Ecm16（也称为Dhr1）相互作用。Ecm16也是在A2位点切割所必需的。我们在ECM16中鉴定出了一些突变，这些突变抑制了bud23Δ突变体的生长和A2切割缺陷。RNA解旋酶通常需要蛋白质辅助因子来提供底物特异性。我们利用酵母（酿酒酵母）双杂交分析来定位Bud23在Ecm16上的结合位点。尽管这些因子之间存在物理和功能上的相互作用，但通过双杂交分析检测到的破坏这种相互作用的突变并未表现出生长缺陷。我们之前在UTP2和UTP14中鉴定出了抑制bud23Δ的突变。我们认为蛋白质相互作用网络可能掩盖了我们通过双杂交分析所定义的相互作用的丧失。Ecm16中基序I的一个突变预计会损害其水解ATP的能力，导致Bud23在含有Ecm16的约45S颗粒中积累。因此，Bud23在Ecm16发挥功能时进入前40S途径。
