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Tandem array–based expression screens identify host mRNA targets of virus-encoded microRNAs


MicroRNAs (miRNAs) are short noncoding RNAs of cellular1 and viral origin2,3,4,5,6,7 that post-transcriptionally regulate gene expression through imperfect base pairing to their mRNA targets. Because the recognition sequences of miRNAs for their targets are short and may be discontinuous, bioinformatic prediction of targets is difficult. Here we present an approach to the experimental identification of the mRNA targets of miRNAs encoded by the Kaposi's sarcoma–associated herpesvirus (KSHV). KSHV encodes 17 miRNAs, derived from 12 pre-miRNAs expressed from a single locus during viral latency2,5,6,7,8,9,10. We conducted multiple screens that examine small changes in transcript abundance under different conditions of miRNA expression or inhibition and then searched the identified transcripts for seed sequence matches. Using this strategy, we identified BCLAF1, encoding Bcl2-associated factor, as a target for miR-K5, and further analysis revealed that several other KSHV miRNAs also target this gene product. Our results support that this type of expression profiling provides a potentially general approach to the identification of miRNA targets.

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Figure 1: Candidate mRNA targets of KSHV miR-K5.
Figure 2: BCLAF1 protein and reporter gene expression analysis.
Figure 3: BCLAF1 protein and reporter gene expression analysis with various KSHV miRNAs.
Figure 4: Apoptosis assays in HUVEC with various miRNAs.
Figure 5: Inhibition of miRNAs and BCLAF1 expression.

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We are grateful to S. Chandriani (University of California, San Francisco) for sharing the HUVEC expression data, expertise and advice. We thank A. Goga (UCSF) and P. Lengyel (UCSF) for sharing a custom gateway cloning vector. The recombinant KSHV virus was a gift from J. Vieira (University of Washington). The infected SLK cell line was generated by J. Myoung (UCSF). PUMAdb is supported by US National Institutes of Health grant P50 GM071508. J.M.Z. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1793). D.G. is an investigator of the Howard Hughes Medical Institute.

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D.G., C.S.S. and J.M.Z. designed the experiments. C.S.S. generated the retroviruses, stable cell lines and performed RNA blots. J.M.Z. conducted the remaining experiments shown in the figures. D.G. directed and supervised the experimental progress. J.M.Z. and D.G. wrote the manuscript.

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Correspondence to Don Ganem.

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Ziegelbauer, J., Sullivan, C. & Ganem, D. Tandem array–based expression screens identify host mRNA targets of virus-encoded microRNAs. Nat Genet 41, 130–134 (2009).

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