In Vivo Reporter System for miRNA-Mediated RNA Silencing.

Abstract

Argonaute proteins play a central role in the evolutionarily conserved mechanisms of RNA silencing. Programmed by a variety of small RNAs, including miRNAs, they recognize their target nucleic acids and modulate gene expression by various means. Argonaute proteins are large complex molecules. Therefore, to better understand the mechanisms they use to regulate gene expression, it is necessary to identify regions of them bearing functional importance (protein-protein interaction surfaces, acceptor sites of posttranslational modifications, etc.). Identification of these regions can be performed using a variety of mutant screens. Here we describe a transient reporter assay system, which is suitable to carry out rapid functional assessment of mutant Argonaute molecules before proceeding to their more detailed biochemical characterization.