High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

Dicer is a multi-functional protein that plays a critical role in regulating several fundamental cellular processes, including RNA interference, genome integrity, development, and antiviral immunity (for recent reviews see [1, 2] ). It is a type III endoribonuclease (RNAse) that contributes to RNA interference by processing specific hairpin structures and long double-stranded RNAs into microRNAs (miR-NAs) and small interfering RNAs (siRNAs), respectively. In the miRNA pathway, Dicer is responsible for cleaving the precursor-miRNA (pre-miRNA) into a~22-nucleotide (nt) duplex, from which one strand will be loaded into the RNA-induced silencing complex (RISC) to repress mRNA translation. Currently, it is thought that Dicer produces almost all miRNAs in human cells (~2000), which together control over 60% of protein-coding genes [3] [4] [5] [6] [7] . The pre-miRNA processing activity of Dicer is known to be highly regulated, and this likely contributes to precisely control miRNA levels and thereby determine cell behavior and cell fate. The cleavage activity is regulated by protein cofactors, such as the TAR RNA Binding Protein (TRBP) and the Protein ACTivator of the interferon-induced protein kinase (PACT), as well as by several RNA-binding proteins known to act either as activators or inhibitors (reviewed in [8] ). Moreover, high-throughput studies have identified additional non-coding RNAs that are likely processed by Dicer [9] as well as several pre-miRNA binding proteins that may regulate its cleavage activity [10] [11] [12] . At this time, there is a need to integrate the available data into a coherent and detailed mechanistic understanding of Dicer's activities and regulation via in vitro biochemical, biophysical and structural studies. However, for such research investigations to be carried out effectively, large quantities of highly pure and active recombinant proteins are needed.