First step in pre-miRNAs processing by human Dicer.
OBJECTIVE
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.


CONCLUSIONS
The differential first-nick is observed in cleavage reactions only when stem-loops are substrates for hDicer.
Flores-Jasso
Carlos Fabián
CF
Departamento de Biología Celular, Instituto de Fisiología Celular, UNAM Apartado Postal 70-600, Circuito exterior s/n Ciudad Universitaria, Delegación Coyoacan, CP 04510, México DF.
Arenas-Huertero
Catalina
C
Reyes
Jose Luis
JL
Contreras-Cubas
Cecilia
C
Covarrubias
Alejandra
A
Vaca
Luis
L
D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*
D035683:MicroRNAs; D011994:Recombinant Proteins; D043244:Ribonuclease III