Dynamic association–dissociation and harboring of endogenous mRNAs in stress granules
Dynamic association–dissociation and harboring of endogenous mRNAs in stress granules
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DOI：
10.1242/jcs.090951
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发表时间：
2011-12
影响因子：
4
通讯作者：
Junwei Zhang;K. Okabe;T. Tani;T. Funatsu
中科院分区：
文献类型：
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作者：
Junwei Zhang;K. Okabe;T. Tani;T. Funatsu
作为对环境胁迫的响应，细胞质中的mRNAs聚集形成应激颗粒(SGS)。目前主要利用蛋白质标记物间接研究SGS，但内源mRNAs在SGS中的实时行为仍不确定。在这里，我们使用线性反义2‘-O-甲基RNA探针在活的哺乳动物细胞中观察到内源性Poly(A)+mRNAs。在亚砷酸盐胁迫的细胞中，内源性mRNAs聚集在与TIA-1-GFP标记的SGS共定位的颗粒中。此外，利用光漂白后的荧光恢复对mRNA动态分析表明，在SGS中，大约三分之一的内源mRNAs是静止的，另外三分之一是扩散的，剩下的三分之一处于与SGS结合和解离之间的平衡状态，时间常数约为300秒。这些mRNAs的动态特性与应激持续时间和微管完整性无关。用反义2‘-O-甲基RNA探针标记的FOS mRNA也有类似的特征。我们的结果揭示了内源性mRNAs的行为，表明SGS是未翻译的Poly(A)+mRNAs的动态港湾。
In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)+ mRNAs in living mammalian cells using a linear antisense 2′-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1–GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2′-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs.