The dynamics of pre-mRNAs and poly(A)+ RNA at speckles in living cells revealed by iFRAP studies

被引:23
作者
Ishihama, Yo [2 ]
Tadakuma, Hisashi [2 ]
Tani, Tokio [3 ]
Funatsu, Takashi [1 ,4 ]
机构
[1] Univ Tokyo, Grad Sch Pharmaceut Sci, Lab Bio Analyt Chem, Bunkyo Ku, Tokyo 1130033, Japan
[2] Waseda Univ, Grad Sch Sci & Engn, Shinjuku Ku, Tokyo 1698555, Japan
[3] Kumamoto Univ, Grad Sch Sci & Technol, Dept Biol Sci, Kumamoto 8608555, Japan
[4] Univ Tokyo, NanoBio Integrat, Tokyo 1130033, Japan
关键词
mRNA; splicing; speckle; iFRAP; live cell imaging;
D O I
10.1016/j.yexcr.2007.10.023
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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 halftime 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. (C) 2007 Elsevier Inc. All rights reserved.
引用
收藏
页码:748 / 762
页数:15
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