期刊
MOLECULAR AND CELLULAR BIOLOGY
卷 29, 期 12, 页码 3243-3254出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.00360-09
关键词
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资金
- CREST
- JST
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan
- Ministry of Education, Culture, Sports, Science and Technology
- Special Coordination Funds for Promoting Science and Technology (SCF)
- Grants-in-Aid for Scientific Research [20112007] Funding Source: KAKEN
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.
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