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

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

Communication between levels of transcriptional control improves robustness and adaptivity

Regulation of eukaryotic gene expression depends on groups of related proteins acting at the levels of chromatin organization, transcriptional initiation, RNA processing, and nuclear transport. However, a unified understanding of how these different levels of transcriptional control interact has been lacking. Here, we combine genome-wide protein–DNA binding data from multiple sources to infer the connections between functional groups of regulators in Saccharomyces cerevisiae. Our resulting transcriptional network uncovers novel biological relationships; supporting experiments confirm new associations between actively transcribed genes and Sir2 and Esc1, two proteins normally linked to silencing chromatin. Analysis of the regulatory network also reveals an elegant architecture for transcriptional control. Using communication theory, we show that most protein regulators prefer to form modules within their functional class, whereas essential proteins maintain the sparse connections between different classes. Moreover, we provide evidence that communication between different regulatory groups improves the robustness and adaptivity of the cell

Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

The localization of active genes to the nuclear periphery is regulated through the cell cycle by Cdk1 phosphorylation of a single nuclear pore protein

Developmentally induced changes in transcriptional program alter spatial organization across chromosomes

Although the spatial location of genes within the nucleus has been implicated in their transcriptional status, little is known about the dynamics of gene location that accompany large-scale changes in gene expression. The mating of haploid yeast Saccharomyces cerevisiae is accompanied by a large-scale change in transcription and developmental program. We examined changes in nuclear organization that accompany stimulus by the mating pheromone α factor and found that most α-factor-induced genes become associated with components of the nuclear envelope. The myosin-like protein Mlp1, which has been implicated in mRNA export, was further shown to exhibit RNA dependence in its association with α-factor-induced genes. High-resolution mapping of association of chromosome III with Mlp1 revealed α-factor-dependent determinants of nuclear pore association, including origins of replication, specific intergenic regions, and the 3′ ends of transcriptionally activated genes. Taken together, these results reveal RNA- and DNA-dependent determinants of nuclear organization as well as a detailed picture of how an entire chromosome alters its spatial conformation in response to a developmental cue

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes

Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors

Hmt1 is the major type I arginine methyltransferase in the yeast Saccharomyces cerevisiae and facilitates the nucleocytoplasmic transport of mRNA-binding proteins through their methylation. Here we demonstrate that Hmt1 is recruited during the beginning of the transcriptional elongation process. Hmt1 methylates Yra1 and Hrp1, two mRNA-binding proteins important for mRNA processing and export. Moreover, loss of Hmt1 affects interactions between mRNA-binding proteins and Tho2, a component of the TREX (transcription/export) complex that is important for transcriptional elongation and recruitment of mRNA export factors. Furthermore, RNA in situ hybridization analysis demonstrates that loss of Hmt1 results in slowed release of HSP104 mRNA from the sites of transcription. Genome-wide location analysis shows that Hmt1 is bound to specific functional gene classes, many of which are also bound by Tho2 and other mRNA-processing factors. These data suggest a model whereby Hmt1 affects transcriptional elongation and, as a result, influences recruitment of RNA-processing factors

Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements

Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e., goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not overinterpreted (the tools are applicable to linear or nonlinear SDEs calibrated from nonstationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single <i>ARG</i>3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT tracking applications

Model of <i>BBC1</i> mRNP association with actin patches.

<p>(A) <i>BBC1</i> can be found in a diffusive state (large green circle) and a confined or stationary state (small green circle) in association with the endocytosing actin patch. Myo3 is represented by a blue rod and actin is colored in red. (B) Deletion of <i>MYO3</i> (<i>myo3Δ</i>) leads to decreased association of <i>BBC1</i> with the actin patch and a decrease in the proportion of <i>BBC1</i> mRNPs in the stationary state (large downward arrow). (C) Disruption of F-actin via addition of latrunculin B causes a decrease in the proportion of <i>BBC1</i> mRNPs at the actin patch (large downward arrow) and an increase in confinement size (mid-sized green circle). Average diffusion coefficients and confinement sizes are displayed.</p

2D dynamics of actin patch mRNA localization.

<p>(A) Montage of a time-lapse video of <i>LAS17:MS2</i> (green) localization in a haploid, early S phase cell with actin patch accumulation at the presumptive bud indicated by FM4–64 staining (red). Scale bar is 2 µm. (B) The trajectory of the mRNA particle in panel (A) is shown on a grayscale image of the first timepoint (0 s) with black-white inversion. Time is encoded by color in the heatmap. (C) Montage of a time-lapse video of <i>LAS17:MS2</i>. Scale bar is 5 µm. Brightness of the latter timepoints in the montage was adjusted relative to the earliest timepoints for display purposes in order to compensate for photobleaching. (D) The trajectory of the mRNA particle from panel (C) is displayed as in panel (B). (E) Change in the diffusion coefficient of the mRNA particle in panels (C) and (D) over time. The diffusion coefficient for short time lags (τ = 4) is graphed with time encoded by color as in panel (D). D was extracted from 4 non-overlapping segments of the trajectory of length 8.992 s, or 16 timepoints. The highest estimated D value, 0.00138 µm²/s, leads to an approximate standard deviation of 5.63×10⁻³ µm²/s <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031912#pone.0031912-Qian1" target="_blank">[135]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031912#pone.0031912-Vrljic1" target="_blank">[136]</a>.</p

Myo3 associates with RNAs encoding key regulators of actin nucleation at actin patches.

<p>(A) Identification of mRNAs associated with motor proteins in <i>S. cerevisiae</i>. Cells encoding motor proteins C-terminally tagged with 9Myc (shown) or EGFP were treated with latrunculin B (top) or fixed with formaldehyde (bottom). Cells were lysed and the motor proteins associated with mRNA cargoes were immunopurified using antibodies coupled to magnetic beads. Enriched mRNAs were eluted from the magnetic beads and, in the case of formaldehyde treatment, decrosslinked before labeling with cyanine dyes. Enriched mRNAs and mRNAs extracted from lysate were comparatively hybridized to yeast DNA oligonucleotide microarrays. (B) and (C) Distribution of the average log₂ Cy5/Cy3 fluorescence ratios from four independent IPs and microarray hybridizations for identification of mRNAs associated with Myo3p in the presence of formaldehyde crosslinker (B) or after cells were incubated with latrunculin B (C). The insets show the mRNAs significantly enriched in Myo3p IPs after subtraction of mRNAs found in mock IPs. mRNAs encoding factors involved in endocytosis at actin patches are labeled in red. (D) Schematic of the steps of endocytic internalization at actin patches and the relative timing of arrival of proteins involved in the regulation and formation of the endosome. Proteins encoded by mRNAs associated with Myo3p are colored in red as in parts (B) and (C).</p