Original Article

First step in pre-miRNAs processing by human Dicer

Carlos Fabián Flores-jasso, Catalina Arenas-huertero, Jose Luis Reyes, Cecilia Contreras-cubas, Alejandra Covarrubias, Luis Vaca
DOI: 10.1038/aps.2009.108

Abstract

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.
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