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.
For each of them, direct interactions between 3'UTR and EBV miRNAs were confirmed by luciferase assay but no functional assay has been performed so far (Table 2).Table 2 Validated targets from high-throughput studies. Shown are cellular targets identified by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP), High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) or RNA immunoprecipitation and microarray (RIP-Chip) techniques. Validation of these targets was done using luciferase reporter assays.
We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs.Table S6: High confidence miRNA-interaction sites in 3'UTRs.We identified an additional 2,972 CDS miRNA-interaction sites present in at least two libraries, 89.7% of which could be assigned to a miRNA (Table S7).
