Systematic Analysis of Viral and Cellular MicroRNA Targets in Cells Latently Infected with Human γ-Herpesviruses by RISC Immunoprecipitation Assay

• Published in: Cell host & microbe 2010-04, Vol.7 (4), p.324-334
• Main Authors: Dölken, Lars, Malterer, Georg, Erhard, Florian, Kothe, Sheila, Friedel, Caroline C., Suffert, Guillaume, Marcinowski, Lisa, Motsch, Natalie, Barth, Stephanie, Beitzinger, Michaela, Lieber, Diana, Bailer, Susanne M., Hoffmann, Reinhard, Ruzsics, Zsolt, Kremmer, Elisabeth, Pfeffer, Sébastien, Zimmer, Ralf, Koszinowski, Ulrich H., Grässer, Friedrich, Meister, Gunter, Haas, Jürgen
• Format: Article
• Language: English
• Subjects: Biochemistry, Molecular Biology; Cell Line; Cells, Cultured; Chromatin Immunoprecipitation; Chromatin Immunoprecipitation - methods; Gene Expression Regulation; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Herpesvirus 4, Human - genetics; Herpesvirus 8, Human; Herpesvirus 8, Human - genetics; Host-Pathogen Interactions; Humans; Life Sciences; Microarray Analysis; MICROBIO; MicroRNAs; MicroRNAs - genetics; MicroRNAs - metabolism; RNA, Viral; RNA, Viral - metabolism; Virology; Virology - methods
• ISSN: 1931-3128; 1934-6069
• DOI: 10.1016/j.chom.2010.03.008

The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3′UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function. ► RIP-Chip identified targets of KSHV and EBV (>150) and cellular (>2300) miRNAs ► Predicted binding sites for miRNAs were overrepresented in 3′UTRs and coding regions ► Viral miRNAs preferentially target cellular genes induced in infected cells ► RIP-Chip analyses offer a quantitative estimate of miRNA function