Xing Li, Niu Meijuan, Zhao Xia, Kleiman Lawrence
Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada.
Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada.
Biochim Biophys Acta. 2014 Jul;1840(7):2234-43. doi: 10.1016/j.bbagen.2014.04.003. Epub 2014 Apr 12.
RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.
The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A-RNA interaction and RNA helicase A-stimulated viral RNA processes.
Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA(Lys3) annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A-HIV-1 RNA interaction in the cells.
The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.
The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.
RNA解旋酶A调节多种RNA代谢过程，包括HIV-1复制，并且在N端含有两个双链RNA结合结构域（dsRBD1和dsRBD2）。每个dsRBD包含两个不变的赖氨酸残基，这两个残基对于分离的dsRBD与RNA的结合至关重要。然而，这些保守的赖氨酸残基在具有酶活性的全长RNA解旋酶A的背景下，无论是在体外还是在细胞中，其作用都未得到测试。
在全长RNA解旋酶A的背景下，将每个或两个dsRBD中的保守赖氨酸残基替换为丙氨酸。从哺乳动物细胞中纯化突变型RNA解旋酶A。这些突变的影响通过体外RNA结合和解旋来评估，或者在细胞中通过RNA解旋酶A-RNA相互作用和RNA解旋酶A刺激的病毒RNA过程来评估HIV-1产生情况。
出乎意料的是，在一个或两个dsRBD中用丙氨酸替换赖氨酸残基并不妨碍纯化的全长RNA解旋酶A在体外结合和解旋双链RNA。然而，这些突变有效地抑制了RNA解旋酶A刺激的HIV-1 RNA代谢，包括病毒mRNA的积累以及tRNA(Lys3)与病毒RNA的退火。此外，这些突变也不妨碍RNA解旋酶A在体外与HIV-1 RNA结合，但显著降低了细胞中RNA解旋酶A与HIV-1 RNA的相互作用。
dsRBD的保守赖氨酸残基在RNA解旋酶A促进HIV-1产生中起关键作用。
dsRBD的保守赖氨酸残基是RNA解旋酶A在细胞中与底物RNA相互作用的关键，但在体外并非如此。
