Functional association of the Microprocessor complex with the spliceosome.
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.
Kataoka
Naoyuki
N
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. kataoka.mtt@mri.tmd.ac.jp
Fujita
Megumi
M
Ohno
Mutsuhito
M
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D046911:Macromolecular Substances; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008954:Models, Biological; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D043244:Ribonuclease III / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; C485471:EML2 protein, human; D046911:Macromolecular Substances; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D012322:RNA Precursors; C517918:DROSHA protein, human; D043244:Ribonuclease III