Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

被引：15
|
作者：
Casolari, Jason M. [1 ,2 ]
Thompson, Michael A. [3 ]
Salzman, Julia [1 ,2 ,4 ]
Champion, Lowry M. [1 ,2 ]
Moerner, W. E. [3 ]
Brown, Patrick O. [1 ,2 ]
机构：
[1] Stanford Univ, Dept Biochem, Sch Med, Stanford, CA 94305 USA
[2] Stanford Univ, Howard Hughes Med Inst, Sch Med, Stanford, CA 94305 USA
[3] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[4] Stanford Univ, Dept Stat, Stanford, CA 94305 USA
来源：
PLOS ONE | 2012年 / 7卷 / 02期
基金：
美国国家卫生研究院; 美国国家科学基金会;
关键词：
POINT-SPREAD FUNCTION; CLASS-V MYOSIN; GENOMIC INTEGRATION METHOD; SINGLE-PARTICLE TRACKING; ACTIN-PATCH MOVEMENT; SACCHAROMYCES-CEREVISIAE; BUDDING YEAST; BINDING PROTEIN; GENE-EXPRESSION; XENOPUS-OOCYTES;
D O I：
10.1371/journal.pone.0031912
中图分类号：
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
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 her e 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.
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页数：20
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