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.
Keywords: Argonaute; Dicer processivity; Precursor miRNA; RISC loading; Target mRNA; miRNA.