Each method has inherent advantages and limitations, but the combination of these approaches have been successful in identifying many miRNA targets (Table 1). Table 1 List of experimentally validated cellular targets of KSHV- and EBV- encoded miRNAs. The normal cellular functions of these proteins and the effect of their knockdown during viral infection are also listed. The targets that are underlined are repressed by both KSHV- and EBV-encoded miRNAs.
Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites.Clusters with seed matches to expressed miRNAs were considered candidate target sites. Of the clusters mapping to human 3'UTRs, 69% (BC-1) and 70% (BC-3) had seed matches to expressed miRNAs (Tables S6).Table S6A BC-1 derived 3'UTR clusters with seed matches to expressed miRNAs.
Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3.
