Ku70作为RNA解旋酶调节miR-124的加工成熟及神经细胞的分化
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1) 北京基础医学研究所,北京 100850;2) 北京微生物流行病研究所,北京 100071

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基金项目:

国家自然科学基金(31370794, 31601055),国家杰出青年基金(81925025),国家自然科学基金创新研究群体(81621005) 和中国医学 科学院医学与健康科技创新工程(2019-I2M-5-049) 资助项目。


Ku70 Functions as an RNA Helicase to Regulate miR-124 Maturation and Neuronal Cell Differentiation
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Affiliation:

1) Beijing Institute of Basic Medical Sciences, Beijing 100850, China;2) Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China

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This work was supported by grants from The National Natural Science Foundation of China (31370794, 31601055), the National Science Fund for Distinguished Young Scholar (81925025), the Innovative Research Group from the NSFC (81621005) , and the Innovation Fund for Medical Sciences from the Chinese Academy of Medical Sciences (2019-I2M-5-049).

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    摘要:

    目的 Ku70蛋白主要通过其DNA结合特性参与双链DNA断裂(DSB)的非同源端连接(NHEJ)修复,有报道称其具有RNA结合功能,本文探索Ku70是否具有RNA解旋酶活性并影响miRNA加工成熟。方法 利用RNA免疫共沉淀(RIP)测序结合生物信息学分析Ku蛋白结合的RNA;蛋白质印迹法(Western blot,WB)结合定量反转录PCR(qRT-PCR)检测Ku蛋白与miRNAs的表达关系;生物膜干涉技术(BLI)实验分析Ku蛋白与RNA的结合能力;电泳迁移率变动分析(EMSA)实验确定Ku70及Ku80的RNA解旋酶活性;形态学检测结合WB分析Ku70调节miR-124引起的神经细胞功能变化;免疫荧光结合形态学分析寨卡病毒(ZIKV)感染后Ku70及miR-124的变化与神经元分化关联。结果 研究发现,Ku70蛋白具有RNA解旋酶活性,并通过其RNA解旋酶活性影响miRNA加工成熟。Ku70缺失引起许多miRNAs上调,其中包括神经细胞特异的miR-124。在人神经前体细胞(hNPCs)和人神经母细胞瘤细胞(SH-SY5Y)中敲低Ku70可促进 miR-124的成熟,从而导致上述细胞向神经元分化。本文进一步发现,ZIKV感染影响了Ku70及miR-124的表达,导致细胞形态的分化。结论 本研究揭示了Ku70的一种新功能,即Ku70有可能参与miRNA的成熟调控和神经细胞的分化,并且可能是ZIKV病毒致小头症的原因之一。

    Abstract:

    Objective Human Ku70 protein mainly involves the non-homologous end joining (NHEJ) repair of double-stranded DNA breaks (DSB) through its DNA-binding properties, and it is recently reported having an RNA-binding ability. This paper is to explore whether Ku70 has RNA helicase activity and affects miRNA maturation.Methods RNAs bound to Ku protein were analyzed by RNA immunoprecipitation sequencing (RIP-seq) and bioinfomatic anaylsis. The expression relationship between Ku protein and miRNAs was verified by Western blot (WB) and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays. Binding ability of Ku protein to the RNAs was tested by biolayer interferometry (BLI) assay. RNA helicase activity of Ku protein was identified with EMSA assay. The effect of Ku70 regulated miR-124 on neuronal differentiation was performed by morphology analysis, WB and immunofluorescence assays with or without Zika virus (ZIKV) infection.Results We revealed that the Ku70 protein had RNA helicase activity and affected miRNA maturation. Deficiency of Ku70 led to the up-regulation of a large number of mature miRNAs, especially neuronal specific miRNAs like miR-124. The knockdown of Ku70 promoted neuronal differentiation in human neural progenitor cells (hNPCs) and SH-SY5Y cells by boosting miR-124 maturation. Importantly, ZIKV infection reduced the expression of Ku70 whereas increased expression of miR-124 in hNPCs, and led to morphologically neuronal differentiation.Conclusion Our study revealed a novel function of Ku70 as an RNA helicase and regulating miRNA maturation. The reduced expression of Ku70 with ZIKV infection increased the expression of miR-124 and led to the premature differentiation of embryonic neural progenitor cells, which might be one of the causes of microcephaly.

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黄皑雪,李睿婷,赵越超,李洁,李慧,丁学锋,王琳,肖参,刘雪梅,秦成峰,邵宁生. Ku70作为RNA解旋酶调节miR-124的加工成熟及神经细胞的分化[J].生物化学与生物物理进展,2024,51(6):1418-1433

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  • 收稿日期:2023-11-20
  • 最后修改日期:2024-05-29
  • 接受日期:2024-01-18
  • 在线发布日期: 2024-06-25
  • 出版日期: 2024-06-20