Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2132328 | Experimental Cell Research | 2008 | 15 Pages |
Abstract
Speckles are subnuclear domains where pre-mRNA splicing factors accumulate in the interchromatin space. To investigate the dynamics of mRNAs at speckles, fluorescently labeled Drosophila Fushitarazu (ftz) pre-mRNAs were microinjected into the nuclei of Cos7 cells and the dissociation kinetics of pre-mRNAs from speckles was analyzed using photobleaching techniques. The microinjected ftz pre-mRNAs accumulated in speckles in an intron-dependent manner and were spliced and exported to the cytoplasm with a half-time of about 10Â min. Dissociation of the accumulated pre-mRNAs in speckles exhibited rapid diffusion and slow-dissociation of about 100Â s. The slow-dissociation required metabolic energy of ATP. Two types of splice-defective mutated mRNAs dissociated from the speckle with a time constant similar to that of wild-type mRNA, indicating that slow-dissociation was not coupled to the splicing reaction. Furthermore, some pre-mRNAs shuttled between speckles and nucleoplasm, suggesting that pre-mRNAs repeatedly associated with and dissociated from speckles until introns were removed. Next, endogenous poly(A)+ RNA was visualized by injecting Cy3-labeled 2â²O-methyl oligo(U)22 probes. Some poly(A)+ RNA distributed diffusely within the nucleus, but some of them accumulated in speckles and dissociated at time constant of about 100Â s.
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Authors
Yo Ishihama, Hisashi Tadakuma, Tokio Tani, Takashi Funatsu,