DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures
DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures
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DOI：
10.1261/rna.2111310
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发表时间：
2010-08-01
期刊：
影响因子：
4.5
通讯作者：
Guo, Feng
中科院分区：
文献类型：
--
作者：
Faller, Michael;Toso, Daniel;Guo, Feng
DiGeorge critical region 8（DGCR 8）是动物microRNA（miRNAs）成熟的关键区域。在Drosha核酸酶切割miRNA的初级转录物（pri-miRNA）时，DGCR 8蛋白通过目前有争议的机制直接结合并识别pri-miRNA。我们先前的数据表明，DGCR 8在与pri-mir-30 a协同结合后三聚化。然而，另一项研究提出了一种模型，其中DGCR 8分子使用其两个双链RNA结合结构域接触一个或两个pri-miRNA分子。在这里，我们使用生物化学和结构方法广泛表征了DGCR 8和pri-miRNAs之间的相互作用。首先，在DGCR 8与pri-mir-30 a的结合和体外pri-miRNA加工速率之间观察到强相关性。第二，我们表明，高结合协同性允许DGCR 8区分pri-miRNAs与非特异性竞争者，其解离常数存在细微差异。DGCR 8与pri-miRNA的高度协同结合是由pri-miRNA上更高级结构的形成介导的，最可能是DGCR 8二聚体的三聚体。这些性质并不限于其与pri-mir-30 a的相互作用。此外，DGCR 8的两亲性C-末端螺旋对于初级-miRNA上DGCR 8的三聚化和Drosha对初级-miRNA的切割都是重要的。最后，我们从负染色的DGCR 8-pri-mir-30 a复合物的电子断层扫描分析得到的三维模型直接支持三聚化模型。我们的研究为DGCR 8识别pri-miRNAs提供了分子基础。我们进一步提出DGCR 8-pri-miRNA复合物的高级结构触发Drosha切割pri-miRNA。
DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.