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.
AIM: To investigate the strand preference of the initial cleavage of human pre-miRNAs by humanDicer 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 humanDicer 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.
Authors: Sam Griffiths-Jones; Russell J Grocock; Stijn van Dongen; Alex Bateman; Anton J Enright Journal: Nucleic Acids Res Date: 2006-01-01 Impact factor: 16.971
Authors: Katie A Mitzelfelt; Pattraranee Limphong; Melinda J Choi; Frances D L Kondrat; Shuping Lai; Kurt D Kolander; Wai-Meng Kwok; Qiang Dai; Michael N Grzybowski; Huali Zhang; Graydon M Taylor; Qiang Lui; Mai T Thao; Judith A Hudson; Rita Barresi; Kate Bushby; Heinz Jungbluth; Elizabeth Wraige; Aron M Geurts; Justin L P Benesch; Michael Riedel; Elisabeth S Christians; Alex C Minella; Ivor J Benjamin Journal: J Biol Chem Date: 2016-05-19 Impact factor: 5.157