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

These updated steady-state kinetic values provide insights into the kinetic mechanism of pre-miRNA cleavage by Dicer. Given the k cat /K M of~10 5 M − 1 s − 1 and the very low k cat value of~0.1 s − 1 , the rate-limiting step may be product release as observed with other RNAse III enzymes [38, 45] or possibly the slow formation and/ or conversion of the enzyme-substrate complex into a catalytically productive conformation. Dicer is a complex and dynamic enzyme that is known to interact with several cellular proteins [46] , and there is increasing evidence that conformational changes may be important for its cleavage activity. Free Dicer is known to adopt different conformations, as confirmed here from negative stain TEM studies, and binding of pre-miRNA or double-stranded RNA substrates is linked to structural rearrangements [18, 23, 27, 37] . Furthermore, singlemolecule fluorescence studies have provided evidence for Dicer adopting two different binding modes with a pre-let-7a-1 substrate [47] . More recently, cryo-EM reconstructions of a pre-miRNA/Dicer/TRBP complex captured two distinct conformations of a pre-dicing state, from which transition to the dicing state would require repositioning of the pre-miRNA in the enzyme processing center [30] . Thus, additional studies are needed to better define the detailed kinetic mechanism of Dicer and characterize the role that conformational changes play along the cleavage reaction pathway.