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Lessons from Nature: microRNA-based shRNA libraries

Abstract

Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.

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Figure 1: Structure of shRNAmir.
Figure 2: Schematics of first- and second-generation library vectors.
Figure 3: Mating scheme for pSM2 library clones.

References

  1. Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    CAS  Article  Google Scholar 

  2. Hamilton, A.J.B. D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).

    CAS  Article  Google Scholar 

  3. Hammond, S.M., Bernstein, E., Beach, D. & Hannon, G.J. An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000).

    CAS  Article  Google Scholar 

  4. Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    CAS  Article  Google Scholar 

  5. Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. & Hannon, G.J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001).

    CAS  Article  Google Scholar 

  6. Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P. & Sharp, P.A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191–3197 (1999).

    CAS  Article  Google Scholar 

  7. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    CAS  Article  Google Scholar 

  8. Song, J-J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. Crystal structure of argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    CAS  Article  Google Scholar 

  9. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  Article  Google Scholar 

  10. Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  Article  Google Scholar 

  11. Lee, R.C., Feinbaum, R.L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854 (1993).

    CAS  Article  Google Scholar 

  12. Wightman, B., Burglin, T.R., Gatto, J., Arasu, P. & Ruvkun, G. Negative regulatory sequences in the lin-14 3′-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes Dev. 5, 1813–1824 (1993).

    Article  Google Scholar 

  13. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  14. He, L. & Hannon, G.J. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531 (2004).

    CAS  Article  Google Scholar 

  15. Lee, Y. et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060 (2004).

    CAS  Article  Google Scholar 

  16. Cai, X., Hagedorn, C.H. & Cullen, B.R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966 (2004).

    CAS  Article  Google Scholar 

  17. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    CAS  Article  Google Scholar 

  18. Denli, A.M., Tops, B.B., Plasterk, R.H., Ketting, R.F. & Hannon, G.J. Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004).

    CAS  Article  Google Scholar 

  19. Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    CAS  Article  Google Scholar 

  20. Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

    CAS  Article  Google Scholar 

  21. Gregory, R.I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    CAS  Article  Google Scholar 

  22. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887–901 (2006).

    CAS  Article  Google Scholar 

  23. Yi, R., Qin, Y., Macara, I.G. & Cullen, B.R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003).

    CAS  Article  Google Scholar 

  24. Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    CAS  Article  Google Scholar 

  25. Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23, 227–231 (2005).

    CAS  Article  Google Scholar 

  26. Song, J.J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032 (2003).

    CAS  Article  Google Scholar 

  27. Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).

    CAS  Article  Google Scholar 

  28. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    CAS  Article  Google Scholar 

  29. Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

    CAS  Article  Google Scholar 

  30. Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37, 1281–1288 (2005).

    CAS  Article  Google Scholar 

  31. Westbrook, T.F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005).

    CAS  Article  Google Scholar 

  32. Kim, D. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol. 23, 222–226 (2005).

    CAS  Article  Google Scholar 

  33. Chen, C.Z., Li, L., Lodish, H.F. & Bartel, D.P. MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86 (2004).

    CAS  Article  Google Scholar 

  34. Zeng, Y. & Cullen, B.R. Sequence requirements for micro RNA processing and function in human cells. RNA 9, 112–123 (2003).

    CAS  Article  Google Scholar 

  35. Ghoda, L., Sidney, D., Macrae, M. & Coffino, P. Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol. Cell. Biol. 12, 2178–2185 (1992).

    CAS  Article  Google Scholar 

  36. Stegmeier, F., Hu, G., Rickles, R.J., Hannon, G.J. & Elledge, S.J. A lentiviral microRNA-based system for single copy Pol II regulated RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 102, 13212–13217 (2005).

    CAS  Article  Google Scholar 

  37. Dickins, R.A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37, 1289–1295 (2005).

    CAS  Article  Google Scholar 

  38. Li, M.Z. & Elledge, S.J. MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat. Genet. 37, 311–319 (2005).

    CAS  Article  Google Scholar 

  39. Zender, L. et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125, 1253–1267 (2006).

    CAS  Article  Google Scholar 

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Correspondence to Gregory J Hannon.

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Competing interests

S.J.E. and G.J.H. are consultants for Open Biosystems, and K.C., S.J.E. and G.J.H. receive royalties from the sales of library materials.

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Chang, K., Elledge, S. & Hannon, G. Lessons from Nature: microRNA-based shRNA libraries. Nat Methods 3, 707–714 (2006). https://doi.org/10.1038/nmeth923

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