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 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.
Highlights
MRNA localization is a fundamental and widespread mechanism for the post-transcriptional regulation of gene expression across many cell types and species [1,2]
While soluble motor proteins could be immunopurified in the absence of formaldehyde, their specific association with mRNAs was less consistent in the absence of crosslinking, and much of the motor protein was found to partition to the insoluble fraction
We used a systematic approach to identify the mRNAs associated with the cytoskeletal motor proteins of S. cerevisiae
Summary
MRNA localization is a fundamental and widespread mechanism for the post-transcriptional regulation of gene expression across many cell types and species [1,2]. The localization of messenger ribonucleoprotein (mRNP) complexes to specific subcellular compartments allows for greater spatial and temporal control of gene expression through regulated and localized translation [2,3]. Among the most fully understood examples of dynamic mRNA localization is the transport of the ASH1 mRNA to the bud tip of Saccharomyces cerevisiae to impart asymmetric production of a transcriptional regulator that effects cell fate determination [6,7,8,9]. When the MS2-CP was fused to GFP and expressed in cells harboring the ASH1 chimeric transcript, Bertrand et al were able to visualize the dynamics of the mRNP in live cells [9]. Subsequent genome-wide studies have indicated that more than 20 transcripts associate with Myo and translocate from the mother cell to the budding daughter cell tip, where they, along with ASH1, are believed to be anchored in an actin-dependent manner [6,7,8,13,18,19]
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