Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).

    Article  CAS  Google Scholar 

  2. Pfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nat. Methods 2, 269–276 (2005).

    Article  CAS  Google Scholar 

  3. Sullivan, C. & Ganem, D. MicroRNAs and viral infection. Mol. Cell 20, 3–7 (2005).

    Article  CAS  Google Scholar 

  4. Stern-Ginossar, N. et al. Host immune system gene targeting by a viral miRNA. Science 317, 376–381 (2007).

    Article  CAS  Google Scholar 

  5. Cai, X. et al. Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc. Natl. Acad. Sci. USA 102, 5570–5575 (2005).

    Article  CAS  Google Scholar 

  6. Samols, M.A., Hu, J., Skalsky, R.L. & Renne, R. Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi's sarcoma-associated herpesvirus. J. Virol. 79, 9301–9305 (2005).

    Article  CAS  Google Scholar 

  7. Grundhoff, A., Sullivan, C. & Ganem, D. A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. RNA 12, 733–750 (2006).

    Article  CAS  Google Scholar 

  8. Samols, M. et al. Identification of cellular genes targeted by KSHV-encoded microRNAs. PLoS Pathog. 3, e65 (2007).

    Article  Google Scholar 

  9. Skalsky, R.L. et al. Kaposi's sarcoma-associated herpesvirus encodes an ortholog of miR-155. J. Virol. 81, 12836–12845 (2007).

    Article  CAS  Google Scholar 

  10. Gottwein, E. et al. A viral microRNA functions as an orthologue of cellular miR-155. Nature 450, 1096–1099 (2007).

    Article  CAS  Google Scholar 

  11. Krützfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  Google Scholar 

  12. Giraldez, A.J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).

    Article  CAS  Google Scholar 

  13. Lim, L. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    Article  CAS  Google Scholar 

  14. Rodriguez, A. et al. Requirement of bic/microRNA-155 for normal immune function. Science 316, 608–611 (2007).

    Article  CAS  Google Scholar 

  15. Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10, 544–550 (2004).

    Article  CAS  Google Scholar 

  16. Hutvagner, G., Simard, M.J., Mello, C.C. & Zamore, P.D. Sequence-specific inhibition of small RNA function. PLoS Biol. 2, E98 (2004).

    Article  Google Scholar 

  17. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    Article  CAS  Google Scholar 

  18. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).

    Article  CAS  Google Scholar 

  19. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).

    Article  CAS  Google Scholar 

  20. Gottwein, E., Cai, X. & Cullen, B.R. A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing. J. Virol. 80, 5321–5326 (2006).

    Article  CAS  Google Scholar 

  21. Didiano, D. & Hobert, O. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat. Struct. Mol. Biol. 13, 849–851 (2006).

    Article  CAS  Google Scholar 

  22. Reinhart, B.J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).

    Article  CAS  Google Scholar 

  23. Yekta, S., Shih, I.H. & Bartel, D.P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004).

    Article  CAS  Google Scholar 

  24. Kasof, G., Goyal, L. & White, E. Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins. Mol. Cell. Biol. 19, 4390–4404 (1999).

    Article  CAS  Google Scholar 

  25. Lewis, B., Burge, C. & Bartel, D. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  Google Scholar 

  26. Jopling, C., Yi, M., Lancaster, A., Lemon, S. & Sarnow, P. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 309, 1577–1581 (2005).

    Article  CAS  Google Scholar 

  27. Offermann, M.K. Kaposi sarcoma herpesvirus-encoded interferon regulator factors. Curr. Top. Microbiol. Immunol. 312, 185–209 (2007).

    CAS  PubMed  Google Scholar 

  28. Oliver, F.J. et al. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J. Biol. Chem. 273, 33533–33539 (1998).

    Article  CAS  Google Scholar 

  29. Vieira, J. & O'Hearn, P.M. Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression. Virology 325, 225–240 (2004).

    Article  CAS  Google Scholar 

  30. Murphy, E., Vanicek, J., Robins, H., Shenk, T. & Levine, A.J. Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: implications for latency. Proc. Natl. Acad. Sci. USA 105, 5453–5458 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Don Ganem.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Table 1 and Supplementary Figures 1–5 (PDF 8493 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/ng.266

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.266

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing