Acta Pharmacologica Sinica (2009) 30: 1177-1185; doi: 10.1038/aps.2009.108

Acta Pharmacologica Sinica (2009) 30: 1177-1185; doi: 10.1038/aps.2009.108

Original Article

First step in pre-miRNAs processing by human Dicer

Carlos Fabián FLORES-JASSO^{1, 3}, Catalina ARENAS-HUERTERO^{2, 4}, Jose Luis REYES², Cecilia CONTRERAS-CUBAS^{1, 3}, Alejandra COVARRUBIAS², Luis VACA^1,*

¹Departamento de Biología Celular, Instituto de Fisiología Celular, UNAM Apartado Postal 70?00, Circuito exterior s/n Ciudad Universitaria, Delegación Coyoacan, CP 04510, México DF; ²Departamento de Biologia Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap Postal 565-A, 62210 Cuernavaca, Mor México; ³Programa de Doctorado en Ciencias Biomédicas, UNAM; ⁴Programa de Maestría y Doctorado en Ciencias Bioquímicas, UNAM

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?/st1:chmetcnv> 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.

Keywords: human Dicer; hsa-pre-miRNA; stem-loop; in vitro; first-nick

We thank P ZAMORE for helpful discussions and for kindly sharing the facilities in his laboratory. We would also like to thank the members of our lab and the Zamore lab for helpful discussions. This work was supported by CONACyT (#42469) and DEGEP.

* To whom correspondence should be addressed.
Email lvaca@ifc.unam.mx
Received 2009-02-18 Accepted 2009-05-27

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