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Abstract
To investigate the strand preference of the initial cleavage of human pre-miRNAs by human Dicer in vitro.
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.
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.
The differential first-nick is observed in cleavage reactions only when stem-loops are substrates for hDicer.