The dynamics of pre-mRNAs and poly(A)+ RNA at speckles in living cells revealed by iFRAP studies.
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 10 min. Dissociation of the accumulated pre-mRNAs in speckles exhibited rapid diffusion and slow-dissociation of about 100 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 2'O-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 100 s.
Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
Yo
Y
Ishihama
Hisashi
H
Tadakuma
Tokio
T
Tani
Takashi
T
Funatsu
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D022003:Cell Nucleus Structures / Q000737:chemistry*; D002522:Chlorocebus aethiops; D029721:Drosophila Proteins / Q000235:genetics; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D051717:Fushi Tarazu Transcription Factors / Q000235:genetics; D007700:Kinetics; D008856:Microscopy, Fluorescence; D012322:RNA Precursors / Q000032:analysis / Q000378:metabolism*; D012326:RNA Splicing; D034443:RNA Transport; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*
D029721:Drosophila Proteins; D051717:Fushi Tarazu Transcription Factors; D012322:RNA Precursors; D012333:RNA, Messenger; C497169:ftz protein, Drosophila