Target mRNA-Driven Biogenesis of Cognate MicroRNAs In Vitro

  • Mainak Bose
  • Suvendra N. Bhattacharyya
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)


miRNAs are 20–22 nucleotide long noncoding RNAs that act as post-transcriptional regulators of gene expression controlling more than half of protein coding genes in humans. Being the critical modulators of the mRNA translation process, biogenesis, function, and turnover of these small RNAs are tightly regulated in cells. We have reported that target mRNAs induce increased biogenesis of cognate miRNAs from pre-miRNAs by increased activity of Ago-associated Dicer endonuclease that processes precursor miRNAs to their mature form. In the current chapter, we discuss how target mRNA-driven RISC loading can be monitored in vitro using affinity-purified miRISC or recombinant AGO2 and DICER1 proteins and scoring the processivity of AGO2-associated DICER1 in vitro.

Key words

miRNA Precursor miRNA Argonaute Dicer processivity RISC loading Target mRNA 


  1. 1.
    Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136:642–655CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139CrossRefPubMedGoogle Scholar
  4. 4.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524CrossRefPubMedGoogle Scholar
  6. 6.
    Maniataki E, Mourelatos Z (2005) A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev 19:2979–2990CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bose M, Bhattacharyya SN (2016) Target-dependent biogenesis of cognate microRNAs in human cells. Nat Commun 7:12200CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ghosh S, Bose M, Ray A, Bhattacharyya SN (2015) Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells. Mol Biol Cell 26:1072–1083CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.RNA Biology Research Laboratory, Molecular Genetics DivisionCSIR-Indian Institute of Chemical BiologyKolkataIndia

Personalised recommendations