Zhu Jieyi, Liu Xin, Anjos Margarida, Correll Carl C, Johnson Arlen W
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
Mol Cell Biol. 2016 Jan 4;36(6):965-78. doi: 10.1128/MCB.00773-15.
In eukaryotic ribosome biogenesis, U3 snoRNA base pairs with the pre-rRNA to promote its processing. However, U3 must be removed to allow folding of the central pseudoknot, a key feature of the small subunit. Previously, we showed that the DEAH/RHA RNA helicase Dhr1 dislodges U3 from the pre-rRNA. DHR1 can be linked to UTP14, encoding an essential protein of the preribosome, through genetic interactions with the rRNA methyltransferase Bud23. Here, we report that Utp14 regulates Dhr1. Mutations within a discrete region of Utp14 reduced interaction with Dhr1 that correlated with reduced function of Utp14. These mutants accumulated Dhr1 and U3 in a pre-40S particle, mimicking a helicase-inactive Dhr1 mutant. This similarity in the phenotypes led us to propose that Utp14 activates Dhr1. Indeed, Utp14 formed a complex with Dhr1 and stimulated its unwinding activity in vitro. Moreover, the utp14 mutants that mimicked a catalytically inactive dhr1 mutant in vivo showed reduced stimulation of unwinding activity in vitro. Dhr1 binding to the preribosome was substantially reduced only when both Utp14 and Bud23 were depleted. Thus, Utp14 is bifunctional; together with Bud23, it is needed for stable interaction of Dhr1 with the preribosome, and Utp14 activates Dhr1 to dislodge U3.
在真核生物核糖体生物合成过程中，U3小核仁RNA（snoRNA）与前体核糖体RNA（pre-rRNA）碱基配对以促进其加工。然而，必须去除U3才能使中央假结折叠，这是小亚基的一个关键特征。此前，我们发现DEAH/RHA RNA解旋酶Dhr1能将U3从前体rRNA上移除。通过与rRNA甲基转移酶Bud23的遗传相互作用，DHR1可以与编码前核糖体必需蛋白的UTP14联系起来。在此，我们报道Utp14对Dhr1具有调控作用。Utp14一个离散区域内的突变减少了与Dhr1的相互作用，这与Utp14功能降低相关。这些突变体在40S前体颗粒中积累了Dhr1和U3，类似于解旋酶失活的Dhr1突变体。这些表型上的相似性使我们推测Utp14激活了Dhr1。事实上，Utp14与Dhr1形成复合物并在体外刺激其解旋活性。此外，在体内模拟催化失活的dhr1突变体的utp14突变体在体外对解旋活性的刺激作用降低。只有当Utp14和Bud23都被耗尽时，Dhr1与前核糖体的结合才会大幅减少。因此,Utp14具有双重功能；与Bud23一起，它是Dhr1与前核糖体稳定相互作用所必需的，并且Utp14激活Dhr1以去除U3。
