Contemporary Ribonomics Methods for Viral microRNA Target Analysis

Numerous cellular processes are regulated by microRNAs (miRNAs), both cellular and viral. Elucidating the targets of miRNAs has become an active area of research. An important method in this field is cross-linking and immunoprecipitation (CLIP), where cultured cells or tissues are UV-irradiated to cross-link protein and nucleic acid, the RNA binding protein of interest is immunoprecipitated, and the RNAs pulled down with the protein are isolated, reverse-transcribed, and analyzed by sequencing. CLIP using antibody against Argonaute (Ago), which binds to both miRNA and mRNA as they interact in RISC, has allowed researchers to uncover a large number of miRNA targets. Coupled with high-throughput sequencing, CLIP has been useful for revealing miRNA targetomes for the γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Variants on the CLIP protocol are described, with the benefits and drawbacks of each. In particular, the most recent methods involving RNA–RNA ligation to join the miRNA and its RNA target have aided in target identification. Lastly, data supporting biologically meaningful interactions between miRNAs and long non-coding RNAs (lncRNAs) are reviewed. In summary, ribonomics-based miRNA targetome analysis has expanded our understanding of miRNA targeting and has provided a rich resource for EBV and KSHV research with respect to pathogenesis and tumorigenesis.