Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR
Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR
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DOI：
10.1261/rna.060269.116
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发表时间：
2017-04-01
期刊：
影响因子：
4.5
通讯作者：
Conn, Graeme L.
中科院分区：
文献类型：
--
作者：
Calderon, Brenda M.;Conn, Graeme L.
双链RNA（dsRNA）激活的蛋白激酶（PKR）检测病毒感染期间产生的dsRNA，并停止细胞蛋白质合成以阻断病毒复制。在没有感染的情况下，基础PKR活性如何被控制尚不清楚，直到最近鉴定出一种潜在的内源性调节因子，细胞非编码RNA 886（nc886）。然而，nc886采用两种不同的构象，其结构细节和潜在的功能差异仍不清楚。在这里，我们分离并分别剖析了每种形式的nc886的功能，以更清楚地定义nc886介导的PKR抑制的分子机制。我们发现，nc886采用两个稳定的，noninterconverting RNA构象，功能上是不等价的互补RNA结构探测和突变分析结合PKR结合和活性测定。一种构象异构体作为有效的抑制剂，而另一种是能够弱激活激酶的假抑制剂。我们映射的nc886区域所需的高亲和力结合和有效抑制PKR的顶端茎环结构中存在的只有一个构象的RNA。这种结构特征不仅对于抑制PKR自身磷酸化至关重要，而且对于其细胞底物真核翻译起始因子2 α亚基的磷酸化也至关重要。不同的活动的nc886构象的鉴定表明一个潜在的机制，在细胞内产生的PKR调节的梯度，并揭示了一种方式，通过这种方式，一个细胞的非编码RNA可以掩盖或目前的结构特征，以PKR抑制。
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem-loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2 alpha subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.