Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin- Associated mRNAs in S. cerevisiae Jason M. Casolari1,2, Michael A. Thompson3, Julia Salzman1,2,4, Lowry M. Champion1,2, W. E. Moerner3, Patrick O. Brown1,2* 1 Department of Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America, 2 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, United States of America, 3 Department of Chemistry, Stanford University, Stanford, California, United States of America, 4 Department of Statistics, Stanford University, Stanford, California, United States of America 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. Citation: Casolari JM, Thompson MA, Salzman J, Champion LM, Moerner WE, et al. (2012) Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae. PLoS ONE 7(2): e31912. doi:10.1371/journal.pone.0031912 Editor: Jonathan S. Weissman, University of California San Francisco/Howard Hughes Medical Institute, United States of America Received January 10, 2012; Accepted January 15, 2012; Published February 16, 2012 Copyright: 2012 Casolari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (R01CA77097 and R01CA111487) to POB and a grant from the National Institute of General Medical Sciences (R01GM085437) to WEM. JMC acknowledges the Jane Coffin Childs Memorial Research Fund for a postdoctoral fellowship and MAT acknowledges support from a National Science Foundation Graduate Research Fellowship and a Bert and DeeDee McMurtry Stanford Graduate Fellowship. POB is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pbrown@stanford.edu Introduction 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]. Indeed, there is increasing evidence that most, if not all, mRNAs are programmed to localize to specific sites in the cell [4,5]. A systematic study in developing Drosophila embryos found evidence of specific subcellular localization for more than 70% of the mRNAs surveyed; in most cases, the localization pattern paralleled that of the encoded proteins [4]. The dynamic localization of mRNAs to discrete cytological locations is critical for such diverse processes as asymmetric cell fate determination, cell motility, and the establish- ment of cellular and organismal polarity [1,2]. 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]. The ASH1 transcript is transported by the type V myosin, Myo4, and other components of the ‘‘locasome,’’ an mRNP complex that also includes the RNA binding protein (RBP), She2, which recognizes the localization elements in the ASH1 transcript, as well as She3, an adapter protein that bridges the interaction between She2 with Myo4 [8,9,10,11,12,13] and which also directly binds RNA [14]. In order to directly observe ASH1 dynamics, Bertrand et al. [9] constructed a chimeric mRNA comprised of a reporter gene, the ASH1 39-UTR, and many copies of a viral (MS2) RNA hairpin that is capable of binding the MS2 viral coat protein (MS2-CP) with nanomolar affinity [15,16,17]. When the MS2-CP was fused to GFP and expressed in cells harboring the ASH1 chimeric transcript, Bertrand et al. were able PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31912