Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.

中文翻译：

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人类Ku蛋白的RNA结合特性分析揭示了它与7SK snRNA和7SK snRNP复合体的蛋白质成分之间的相互作用。

由Ku70和Ku80亚基组成的人Ku异源二聚体蛋白在非同源末端连接DNA修复途径中起着重要作用，作为双链DNA断裂的传感器。Ku还参与许多细胞过程，在某些过程中，它以RNA依赖性方式发挥作用。然而，人类Ku的RNA结合特性尚未得到很好的研究。在这里，我们已经分析了重组Ku异源二聚体与各种结构的RNA的相互作用以及人类Ku70的eCLIP（增强的交联和免疫沉淀）数据。结果，我们提出了对于Ku结合而言优选的共有RNA结构，该结构是在包含GpG序列的末端环附近具有凸起的发夹。7SK snRNA是核糖核蛋白复合物（7SK snRNP）的支架，已知该复合物参与转录调控。我们已经显示，重组Ku特异性结合7SK snRNA内发夹1的富含G的环。而且，Ku蛋白已经与内源性7SK snRNA从HEK 293T细胞中共沉淀，并且7SK snRNP中包括的这些蛋白如HEXIM1，Cdk9和CTIP2。发现Ku和Cdk9的结合不依赖于RNA，而HEXIM1和Ku的共沉淀依赖于完整的7SK snRNA的存在。使用重组HEXIM1和Ku蛋白已证实了后者的结果。共聚焦显微镜还证实了Ku和CTIP2的共定位。这些结果使我们能够提出人类Ku作为7SK snRNP复合体的新组成部分。Ku蛋白已经与内源性7SK snRNA从HEK 293T细胞中共沉淀，并且7SK snRNP中包括的这些蛋白例如HEXIM1，Cdk9和CTIP2。发现Ku和Cdk9的结合不依赖于RNA，而HEXIM1和Ku的共沉淀依赖于完整的7SK snRNA的存在。使用重组HEXIM1和Ku蛋白已证实了后者的结果。共聚焦显微镜还证实了Ku和CTIP2的共定位。这些结果使我们能够提出人类Ku作为7SK snRNP复合体的新组成部分。Ku蛋白已经与内源性7SK snRNA从HEK 293T细胞中共沉淀，并且7SK snRNP中包括的这些蛋白例如HEXIM1，Cdk9和CTIP2。发现Ku和Cdk9的结合不依赖于RNA，而HEXIM1和Ku的共沉淀依赖于完整的7SK snRNA的存在。使用重组HEXIM1和Ku蛋白已证实了后者的结果。共聚焦显微镜还证实了Ku和CTIP2的共定位。这些结果使我们能够提出人类Ku作为7SK snRNP复合体的新组成部分。共聚焦显微镜还证实了Ku和CTIP2的共定位。这些结果使我们能够提出人类Ku作为7SK snRNP复合体的新组成部分。共聚焦显微镜还证实了Ku和CTIP2的共定位。这些结果使我们能够提出人类Ku作为7SK snRNP复合体的新组成部分。