The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo
The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo
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DOI：
10.1128/mcb.17.6.3194
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发表时间：
1997-06-01
影响因子：
5.3
通讯作者：
Darnell, RB
中科院分区：
文献类型：
--
作者：
Buckanovich, RJ;Darnell, RB
Nova-1是一种神经元特异性核RNA结合蛋白，是副肿瘤性眼阵挛肌阵挛共济失调（POMA）的自身抗原，POMA是一种与乳腺癌和运动功能障碍相关的疾病。我们已经确定了在体内Nova-1 RNA配体结合亲和洗脱的RNA选择与蛋白质-RNA免疫沉淀。从类似于10（15）个随机52-mer RNA的池开始，我们鉴定了以高亲和力（K-d类似于2nM）结合Nova-1的长茎环RNA配体。这些RNA的环区含有一个类似于15-bp的富含嘧啶的元件[UCAU（N）（0-2）]（3），这是Nova-1结合所必需的。突变研究确定Nova-1的第三个KH结构域和[UCAU（N）（0-2）]（3）元件是体外结合所必需的，在两个神经元前mRNA中鉴定了共有[UCAU（N）（0-2）]（3）元件，一个编码抑制性甘氨酸受体α 2（GlyR α 2），第二个编码Nova-1本身。Nova-1蛋白在体外以高亲和力和特异性结合这些RNA，并且这种结合可以被POMA抗血清阻断。此外，Nova-1和GlyR α 2前体mRNA与来自脑提取物的Nova-1蛋白特异性共免疫沉淀。因此，Nova-1在体内作为序列特异性核RNA结合蛋白发挥功能; Nova-1和GlyR α 2前体mRNA之间特异性相互作用的破坏可能是POMA中观察到的运动功能障碍的基础。
Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of similar to 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (K-d of similar to 2 nM). The loop region of these RNAs harbors a similar to 15-bp pyrimidine-rich element [UCAU(N)(0-2)](3) which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)](3) element as necessary for in vitro binding, Consensus [UCAU (N)(0-2)](3) elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha 2 (GlyR alpha 2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha 2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha 2 pre-mRNA may underlie the motor dysfunction seen in POMA.