Quantifying Protein-mRNA Interactions in Single Live Cells.
Quantifying Protein-mRNA Interactions in Single Live Cells.
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DOI：
10.1016/j.cell.2015.05.054
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发表时间：
2015-07-02
期刊：
影响因子：
64.5
通讯作者：
Singer RH
中科院分区：
文献类型：
--
作者：
Wu B;Buxbaum AR;Katz ZB;Yoon YJ;Singer RH
特异性结合蛋白对于mRNA的正确时空表达至关重要。为了理解这一过程，需要一种方法来表征单个活细胞中的RNA-蛋白质相互作用的亚细胞分辨率。我们将内源性单个RNA和蛋白质检测与双光子荧光波动分析相结合，以测量活细胞内特定位置处与mRNA结合的蛋白质的平均数量。我们应用这一点来量化已知的zipcode结合蛋白1（ZBP 1）和核糖体与原代成纤维细胞和神经元亚细胞区室中β-肌动蛋白mRNA的结合。ZBP 1-mRNA结合没有发生在细胞核中，与以前的结论相反。与成纤维细胞相比，ZBP 1与β-actin mRNA的相互作用在神经元的核周增强。胞质ZBP 1和核糖体结合的mRNA是反相关的，这取决于它们在细胞中的位置。这些测量支持ZBP 1抑制定位mRNA的翻译直到其从mRNA外周释放，允许核糖体结合的机制。
Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes, to β-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with β-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.
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影响因子：
48
作者：
Mali, Prashant;Esvelt, Kevin M.;Church, George M.
通讯作者：
Church, George M.
DOI：
10.1038/nrm3918
发表时间：
2015-02
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
通讯作者：
Tuschl T
影响因子：
56.9
作者：
Femino, A;Fay, FS;Singer, RH
通讯作者：
Singer, RH
DOI：
10.1126/science.1242939
发表时间：
2014-01-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Buxbaum AR;Wu B;Singer RH
通讯作者：
Singer RH