Zhang Suisheng, Schlott Bernhard, Görlach Matthias, Grosse Frank
Department of Biochemistry, Institute of Molecular Biotechnology, Postfach 100 813, D-07708 Jena, Germany.
Nucleic Acids Res. 2004 Jan 2;32(1):1-10. doi: 10.1093/nar/gkg933. Print 2004.
An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.
通过免疫沉淀和凝胶过滤色谱法在HeLa细胞核提取物中发现，Ku抗原与核DNA解旋酶II（NDH II，也称为RNA解旋酶A，即RHA）存在RNA依赖性关联。Ku抗原和NDH II均与hnRNP复合物相关。二维凝胶电泳显示，Ku抗原与hnRNP C、K、J、H和F的关联最为丰富，但显然与其他蛋白，如hnRNP A1无关。出乎意料的是，由Ku抗原作为DNA结合亚基组成的DNA依赖性蛋白激酶（DNA-PK）以RNA依赖性方式使hnRNP蛋白磷酸化。在RNA存在的情况下，DNA-PK也使重组NDH II磷酸化。RNA结合试验表明，DNA-PK对聚（rG）有偏好性，但对聚（rA）、聚（rC）或聚（rU）没有偏好。DNA-PK的这种RNA结合亲和力可归因于其Ku86亚基。一致地，聚（rG）最强烈地刺激了DNA-PK催化的NDH II磷酸化。RNA干扰研究表明，NDH II表达受抑制会改变hnRNP C的核分布，而使DNA-PK沉默则会改变NDH II和hnRNP C的核内亚分布。这些结果支持这样一种观点，即DNA-PK也可作为一种RNA依赖性蛋白激酶，调节RNA代谢的某些方面，如RNA加工和转运。
