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.
Ishihama
Yo
Y
Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
Tadakuma
Hisashi
H
Tani
Tokio
T
Funatsu
Takashi
T
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