ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.
ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.
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DOI：
10.1093/nar/gkv614
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发表时间：
2015-10-30
影响因子：
14.9
通讯作者：
Wang DO
中科院分区：
文献类型：
--
作者：
Oomoto I;Suzuki-Hirano A;Umeshima H;Han YW;Yanagisawa H;Carlton P;Harada Y;Kengaku M;Okamoto A;Shimogori T;Wang DO
阐明核RNA焦点的动态组织对于理解和操纵这些生理和病理状态下基因表达的功能位点都是重要的。然而，由于缺乏合适的RNA成像方法，此类研究很难在体内建立。在这里，我们描述了一种高分辨率的荧光RNA成像方法，ECHO-LiveFISH，用于标记活着的小鼠和小鸡的内源性核RNA。在体内电穿孔后，激子控制的序列特异性寡核苷酸探针在核仁上发现了集中的内源性28S rRNA和U3 snoRNA，在核斑点上发现了Poly(A)RNA。延时成像显示了这些RNA病灶的稳态稳定性，以及在天然脑组织中聚合酶I抑制时28S rRNA浓度的动态消散。体内RNA标记没有干扰靶RNA的功能，也没有引起明显的细胞毒性或细胞行为的扰动，这证实了这项技术在生理环境中的有效性。
Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.