Advertisement

Cellular Approaches in Investigating Argonaute2-Dependent RNA Silencing

  • Cai Zhang
  • Joonbae Seo
  • Takahisa NakamuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)

Abstract

In mammals, there are four Argonaute (Ago) family proteins that play crucial roles in RNA silencing, a process wherein microRNA (miRNA) mediates inhibition of target mRNA translation. Among the Ago proteins, Argonaute2 (Ago2) uniquely possesses an endoribonuclease (slicer) activity that is critical for the biogenesis of specific miRNAs and mRNA cleavage. This Ago2 slicer activity is required for postnatal development. Despite its important roles, there are still gaps in our understanding of the mechanistic basis of Ago2’s unique functions in vivo due to a limited availability of experimental tools. In order to investigate Ago2’s functions, we generated a new cellular model of Ago2-deficiency in 3T3 mouse embryonic fibroblasts (MEFs). This cell line can be used for investigating general Ago2 functions, but also for further understanding of Ago2’s unique characteristics including the slicer activity, specific amino acid residues, and domains in Ago2 by reconstitution of Ago2 mutants. Here, we describe the methods for establishing Ago2-deficient MEFs and for reconstituting the MEFs with an Ago2 mutant lacking its slicer activity by means of a retrovirus-mediated gene transfer.

Key words

Ago2 3T3 protocol Mouse embryonic fibroblast 

Notes

Acknowledgments

We thank Kazutoshi Murakami, Elise Bernhard, and Vishnupriya Borra for discussions. This work was supported by NIH RO1 DK107530, Digestive Disease Research Core Center in Cincinnati (DK078392), and PRESTO from the Japan Science and Technology Agency.

References

  1. 1.
    Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123(4):631–640. doi: 10.1016/j.cell.2005.10.022 CrossRefPubMedGoogle Scholar
  2. 2.
    Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ (2010) A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465(7298):584–589. doi: 10.1038/nature09092 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Yang JS, Maurin T, Robine N, Rasmussen KD, Jeffrey KL, Chandwani R, Papapetrou EP, Sadelain M, O'Carroll D, Lai EC (2010) Conserved vertebrate mir-451 provides a platform for dicer-independent, Ago2-mediated microRNA biogenesis. Proc Natl Acad Sci U S A 107(34):15163–15168. doi: 10.1073/pnas.1006432107 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    O’Carroll D, Mecklenbrauker I, Das PP, Santana A, Koenig U, Enright AJ, Miska EA, Tarakhovsky A (2007) A slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway. Genes Dev 21(16):1999–2004. doi: 10.1101/gad.1565607 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi: 10.1038/nrg3462 CrossRefPubMedGoogle Scholar
  6. 6.
    Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689):1437–1441. doi: 10.1126/science.1102513 CrossRefPubMedGoogle Scholar
  7. 7.
    Cheng N, Li Y, Han ZG (2013) Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma. Hepatology 57(5):1906–1918. doi: 10.1002/hep.26202 CrossRefPubMedGoogle Scholar
  8. 8.
    Shen J, Xia W, Khotskaya YB, Huo L, Nakanishi K, Lim SO, Du Y, Wang Y, Chang WC, Chen CH, Hsu JL, Wu Y, Lam YC, James BP, Liu X, Liu CG, Patel DJ, Hung MC (2013) EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2. Nature 497(7449):383–387. doi: 10.1038/nature12080 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, Cheloufi S, Ma E, Mane S, Hannon GJ, Lawson ND, Wolfe SA, Giraldez AJ (2010) A novel miRNA processing pathway independent of dicer requires Argonaute2 catalytic activity. Science 328(5986):1694–1698. doi: 10.1126/science.1190809 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Tattikota SG, Rathjen T, McAnulty SJ, Wessels HH, Akerman I, van de Bunt M, Hausser J, Esguerra JL, Musahl A, Pandey AK, You X, Chen W, Herrera PL, Johnson PR, O’Carroll D, Eliasson L, Zavolan M, Gloyn AL, Ferrer J, Shalom-Feuerstein R, Aberdam D, Poy MN (2014) Argonaute2 mediates compensatory expansion of the pancreatic beta cell. Cell Metab 19(1):122–134. doi: 10.1016/j.cmet.2013.11.015 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ma JB, Yuan YR, Meister G, Pei Y, Tuschl T, Patel DJ (2005) Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434(7033):666–670. doi: 10.1038/nature03514 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Horman SR, Janas MM, Litterst C, Wang B, MacRae IJ, Sever MJ, Morrissey DV, Graves P, Luo B, Umesalma S, Qi HH, Miraglia LJ, Novina CD, Orth AP (2013) Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets. Mol Cell 50(3):356–367. doi: 10.1016/j.molcel.2013.03.015 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16(7):421–433. doi: 10.1038/nrg3965 CrossRefPubMedGoogle Scholar
  14. 14.
    Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17:299–313CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Pediatrics, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
  2. 2.Division of EndocrinologyCincinnati Children’s Hospital Medical CenterCincinnatiUSA
  3. 3.Division of Developmental BiologyCincinnati Children’s Hospital Medical CenterCincinnatiUSA

Personalised recommendations