Target mRNA-Driven Biogenesis of Cognate MicroRNAs In Vitro

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)

Abstract

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 

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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

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