Aim: To investigate the strand preference of the initial cleavage of human pre-miRNAs by human Dicer in vitro.Methods: We used a series of in vitro transcribed pre-miRNAs that were radioactively labeled at their 5' or 3' ends in cleavage reactions with recombinant human Dicer or HeLa cytoplasmic S100 extracts. Pre-miRNAs samples were purified by denaturing and native PAGE and only the stem-loop structures were used in the experiments. Products of cleavage reactions were resolved by denaturing PAGE, and scanned by phosphor-imaging.Results: Recombinant hDicer performs a biased first-cleavage in the pre-let-7b and hsa-pre-miR-17 3' strand. This result is recapitulated in HeLa S100 cytoplasmic extracts.Conclusion: The differential first-nick is observed in cleavage reactions only when stem-loops are substrates for hDicer.
Última actualización: 24/10/2016