Yamane Daisuke, Selitsky Sara R, Shimakami Tetsuro, Li You, Zhou Mi, Honda Masao, Sethupathy Praveen, Lemon Stanley M
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Departments of Medicine and Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA.
Nucleic Acids Res. 2017 May 5;45(8):4743-4755. doi: 10.1093/nar/gkw1332.
In addition to suppressing cellular gene expression, certain miRNAs potently facilitate replication of specific positive-strand RNA viruses. miR-122, a pro-viral hepatitis C virus (HCV) host factor, binds and recruits Ago2 to tandem sites (S1 and S2) near the 5΄ end of the HCV genome, stabilizing it and promoting its synthesis. HCV target site selection follows canonical miRNA rules, but how non-templated 3΄ miR-122 modifications impact this unconventional miRNA action is unknown. High-throughput sequencing revealed that a 22 nt miRNA with 3΄G ('22-3΄G') comprised <63% of total miR-122 in human liver, whereas other variants (23-3΄A, 23-3΄U, 21-3΄U) represented 11-17%. All loaded equivalently into Ago2, and when tested individually functioned comparably in suppressing gene expression. In contrast, 23-3΄A and 23-3΄U were more active than 22-3΄G in stabilizing HCV RNA and promoting its replication, whereas 21-3΄U was almost completely inactive. This lack of 21-3΄U HCV host factor activity correlated with reduced recruitment of Ago2 to the HCV S1 site. Additional experiments demonstrated strong preference for guanosine at nt 22 of miR-122. Our findings reveal the importance of non-templated 3΄ miR-122 modifications to its HCV host factor activity, and identify unexpected differences in miRNA requirements for host gene suppression versus RNA virus replication.
除了抑制细胞基因表达外，某些微小RNA（miRNA）还能有效地促进特定正链RNA病毒的复制。miR-122是丙型肝炎病毒（HCV）的一种前病毒宿主因子，它与HCV基因组5΄端附近的串联位点（S1和S2）结合并招募AGO2，使其稳定并促进其合成。HCV靶位点的选择遵循典型的miRNA规则，但非模板化的3΄ miR-122修饰如何影响这种非常规的miRNA作用尚不清楚。高通量测序显示，带有3΄G的22 nt miRNA（“22-3΄G”）在人肝脏中占总miR-122的比例不到63%，而其他变体（23-3΄A、23-3΄U、21-3΄U）占11-17%。所有这些变体都能同等程度地加载到AGO2中，单独测试时在抑制基因表达方面功能相当。相比之下，23-3΄A和23-3΄U在稳定HCV RNA和促进其复制方面比22-3΄G更活跃，而21-3΄U几乎完全没有活性。21-3΄U缺乏HCV宿主因子活性与AGO2向HCV S1位点的募集减少有关。进一步的实验表明，miR-122的第22位核苷酸强烈偏好鸟苷。我们的研究结果揭示了非模板化的3΄ miR-122修饰对其HCV宿主因子活性的重要性，并确定了miRNA在宿主基因抑制和RNA病毒复制方面需求的意外差异。
