Fan Baochang, Sutandy F X Reymond, Syu Guan-Da, Middleton Stefani, Yi Guanghui, Lu Kuan-Yi, Chen Chien-Sheng, Kao C Cheng
From the ‡Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA;
§Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli 32001, Taiwan.
Mol Cell Proteomics. 2015 Nov;14(11):2878-86. doi: 10.1074/mcp.M115.050344. Epub 2015 Sep 1.
Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.
异质性核糖核蛋白K（hnRNP K）与丙型肝炎病毒（HCV）的5'非翻译区结合，是HCV RNA复制所必需的。HCV RNA上的hnRNP K结合位点与肝脏特异性微小RNA miR-122识别的序列重叠。用miR-122探测的包含约17,000种独特人类蛋白质的蛋白质组芯片将hnRNP K鉴定为强结合蛋白之一。体外动力学研究表明，hnRNP K以纳摩尔解离常数结合miR-122，其中miR-122中央和3'部分富含嘧啶的短残基是hnRNP K结合所必需的。在肝肝细胞中，miR-122与hnRNP K形成可共沉淀复合物。对与hnRNP K一起沉淀下来的RNA进行的高通量Illumina DNA测序富集了成熟的miR-122。在人肝细胞中通过小干扰RNA敲低hnRNP K可降低miR-122的水平。这些结果表明，hnRNP K是一种与miR-122结合并影响其积累的细胞蛋白。它在miR-122结合位点附近还能结合HCV RNA的能力提示了miR-122识别HCV RNA的作用。
