Here,we explore the similarities and differences in the functions of miRNA targets from the limited number of experimentally validated miRNA targets and the larger number of targets predicted by recent reports using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation(HITS-CLIP and PAR-CLIP) (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 and microarray(RIP-Chip)techniques. Validation of these targets was done using luciferase reporter assays.
We focused this analysis mostly on 3'UTRs of mRNAs encoding proteins with functions of potential relevance to KSHV biology and pathogenesis (Table S11). Thirty-six combinations of 3'UTR indicators and KSHV miRNAs with PAR-CLIP interactions were tested for regulation. Nine of these interactions were independently also predicted by microarray analysis of the BJAB cell pools expressing miR-K1, miR-K4-3p, or miR-K11 described above (i.e. for miR-K1: NMI, RAD21, BCL11A, RFXAP; for miR-K4-3p: TPD52, GRB2, MCC, YWHAB; for miR-K11: WEE1). Targets were representative of the full range of seed match types observed in the libraries (Tables S6 and S11).
As KSHV putative direct targets we extracted transcripts that were significantly down-regulated significantly in the replicate experiments, and which contained at least one seed-match to one of the KSHV miRNAs. We identified 704 putative direct targets in DG-75 cells (Figure 3B), and 980 putative direct targets in EA.hy926 cells (Figure 3C). A complete list of putative direct targets can be found in Dataset S2 for DG-75 cells and in Dataset S3 for EA.hy926 cells. The overlap between the two datasets contained 153 putative direct targets (Dataset S4).
