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mRNA 与细胞骨架马达蛋白的广泛关联，以及肌球蛋白相关 mRNAs 在酿酒酵母中的鉴定和动态。

Widespread mRNA association with cytoskeletal motor proteins and identification and dynamics of myosin-associated mRNAs in S. cerevisiae.

机构信息

Department of Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America.

出版信息

PLoS One. 2012;7(2):e31912. doi: 10.1371/journal.pone.0031912. Epub 2012 Feb 16.

DOI:10.1371/journal.pone.0031912
PMID:22359641
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3281097/
Abstract

Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe here a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.

摘要

将 mRNA 定位到特定的亚细胞隔室以进行局部翻译是真核生物中转录后调控的基本机制，影响许多（如果不是全部的话）mRNA。我们在这里描述了一种系统的方法，用于鉴定与酿酒酵母细胞骨架马达蛋白相关的 RNA 货物，同时结合内源性标记的 mRNA 的活细胞 3D 超定位显微镜。我们的分析确定了 mRNA 与细胞骨架马达蛋白的广泛关联，包括 Myo3 与编码肌动蛋白分支和内吞作用的关键调节剂（如 WASP 和 WIP）的 mRNA 的关联。使用常规荧光显微镜和来自内源性基因座的 MS2 标记的 mRNA 的表达，我们观察到肌动蛋白斑块核酶 mRNA 强烈偏向于定位到细胞皮层和肌动蛋白斑块，这依赖于 Myo3 和 F-肌动蛋白。使用双螺旋点扩散函数 (DH-PSF) 显微镜，在 50 毫秒曝光时间的活细胞中以 25nm 的空间精度在 x 和 y 方向和 50nm 的 z 方向进行单个 mRNP 的超定位测量，从而可以对 mRNP 动力学进行定量分析。与对照 mRNA 相比，肌动蛋白斑块 mRNA 表现出明显不同的特征扩散系数。此外，F-肌动蛋白的破坏显著扩大了肌动蛋白斑块核酶 mRNA 的 3D 限制半径，为肌动蛋白细胞骨架对 mRNP 动态定位的贡献提供了定量评估。我们的结果为酵母中 mRNA 与细胞骨架马达蛋白的特定关联提供了证据，表明不同的 mRNP 具有不同的特征动力学，并深入了解肌动蛋白斑块核酶 mRNA 定位到肌动蛋白斑块的机制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/3833c9729c3b/pone.0031912.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/159d0f8c7556/pone.0031912.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/d1dde2eecd03/pone.0031912.g002.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/15fc88551de8/pone.0031912.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/4728a4dfc218/pone.0031912.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/2e5149e431a4/pone.0031912.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/3833c9729c3b/pone.0031912.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/159d0f8c7556/pone.0031912.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/d1dde2eecd03/pone.0031912.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/e9287a684433/pone.0031912.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/15fc88551de8/pone.0031912.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/4728a4dfc218/pone.0031912.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/2e5149e431a4/pone.0031912.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/503d/3281097/3833c9729c3b/pone.0031912.g007.jpg

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