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Molecular and Cellular Biology, June 2009, p. 3243-3254, Vol. 29, No. 12
0270-7306/09/$08.00+0     doi:10.1128/MCB.00360-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Association of the Microprocessor Complex with the Spliceosome{triangledown} ,{dagger}

Naoyuki Kataoka,1,2*,{ddagger} Megumi Fujita,1,{ddagger} and Mutsuhito Ohno1*

Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan,1 Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan2

Received 20 March 2009/ Accepted 26 March 2009

The majority of human microRNAs (miRNAs) are located in the introns of other genes (A. Rodriguez, S. Griffiths-Jones, J. L. Ashurst, and A. Bradley, Genome Res. 14:1902-1910, 2004). Based on the discovery that artificial insertion of pre-miRNAs in introns did not hamper mRNA production and that the miRNA-harboring introns were spliced more slowly than the adjacent introns, a model was previously proposed in which Drosha crops the pre-miRNA and the two cropped fragments from the pre-mRNA are subsequently trans spliced (Y. K. Kim and V. N. Kim, EMBO J. 26:775-783, 2007). However, the molecular basis for this model was not elucidated. To analyze the molecular mechanism of intronic miRNA processing, we developed an in vitro system in which both pre-miRNA processing and mRNA splicing are detected simultaneously. Our analysis using this system showed that pre-miRNA cropping from the pre-mRNA could occur kinetically faster than splicing. Glycerol gradient sedimentation experiments revealed that part of the pre-miRNA was cofractionated with the spliceosome. Furthermore, coimmunoprecipitation experiments with an anti-Drosha antibody demonstrated that Drosha was associated not only with the cropping products but also with a Y-shaped branch intron and a Y-shaped splicing intermediate. These results provide a molecular basis for the postulated existence of a pathway in which the Microprocessor complex becomes associated with the spliceosome, pre-miRNA cropping occurs prior to splicing, and trans splicing takes place between the cropped products.

* Corresponding author. Mailing address for Naoyuki Kataoka: Medical Research Institute, Tokyo Medical and Dental University, Room D509, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-4877. Fax: 81-3-5803-5853. E-mail: kataoka.mtt{at}mri.tmd.ac.jp. Mailing address for Mutsuhito Ohno: Institute for Virus Research, Kyoto University, Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-4018. Fax: 81-75-751-3992. E-mail: hitoohno{at}virus.kyoto-u.ac.jp

{triangledown} Published ahead of print on 6 April 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} N.K. and M.F. contributed equally to this work.

Molecular and Cellular Biology, June 2009, p. 3243-3254, Vol. 29, No. 12
0270-7306/09/$08.00+0     doi:10.1128/MCB.00360-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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