A novel role for GSK3β as a modulator of Drosha microprocessor activity and MicroRNA biogenesis.
A novel role for GSK3β as a modulator of Drosha microprocessor activity and MicroRNA biogenesis.
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DOI：
10.1093/nar/gkw938
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发表时间：
2017-03-17
影响因子：
14.9
通讯作者：
Bevan CL
中科院分区：
文献类型：
--
作者：
Fletcher CE;Godfrey JD;Shibakawa A;Bushell M;Bevan CL
microRNA （miR）生物发生的调控是复杂而严格的。在这里，我们发现激酶GSK3β在微处理器水平上是miR生物发生的重要调节剂。GSK3β活性的抑制降低了对pri-miRs的Drosha活性，导致未加工的pri-miRs的积累和pre-miRs和成熟miRs的减少，而不改变miR生物发生蛋白的水平或细胞定位。相反，GSK3β的激活增加了Drosha活性和成熟miR的积累。GSK3β通过促进Drosha:cofactor和Drosha:pri-miR相互作用来实现这一点：它与微处理器中的DGCR8和p72结合，这种作用依赖于RNA的存在。事实上，GSK3β本身可以免疫沉淀pri-miRs，提示可能的rna结合能力。激酶分析确定了GSK3β增强Drosha活性的机制，这需要GSK3β核定位，如在S300和/或S302位点磷酸化；通过增强Drosha活性和与辅因子的关联，以及在phospho-mimic Drosha存在下成熟miRs丰度的增加，证实了这一点。GSK3β抑制后，GSK3β上调miR靶点的水平增加，说明了GSK3β增强miR生物发生的功能意义。这些数据首次将GSK3β与人类miR级联联系起来，强调了GSK3β作为微处理器复合物的一个组成部分，在增加miR生物发生方面具有新的促生物发生作用，具有广泛的功能后果。
Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here, we identify the kinase GSK3β as an important modulator of miR biogenesis at Microprocessor level. Repression of GSK3β activity reduces Drosha activity toward pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisation of miR biogenesis proteins. Conversely, GSK3β activation increases Drosha activity and mature miR accumulation. GSK3β achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent upon presence of RNA. Indeed, GSK3β itself can immunoprecipitate pri-miRs, suggesting possible RNA-binding capacity. Kinase assays identify the mechanism for GSK3β-enhanced Drosha activity, which requires GSK3β nuclear localisation, as phosphorylation of Drosha at S300 and/or S302; confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature miRs in the presence of phospho-mimic Drosha. Functional implications of GSK3β-enhanced miR biogenesis are illustrated by increased levels of GSK3β-upregulated miR targets following GSK3β inhibition. These data, the first to link GSK3β with the miR cascade in humans, highlight a novel pro-biogenesis role for GSK3β in increasing miR biogenesis as a component of the Microprocessor complex with wide-ranging functional consequences.