ASH1 mRNA localizes to the bud tip in Saccharomyces cerevisiae to establish asymmetry of HO expression, important for mating type switching. To visualize real time localization of the mRNA in living yeast cells, green fluorescent protein (GFP) was fused to the RNA-binding protein MS2 to follow a reporter mRNA containing MS2-binding sites. Formation and localization of a GFP particle in the bud required ASH1 3'UTR (untranslated region) sequences. The SHE mutants disrupt RNA and particle localization and SHE 2 and 3 mutants inhibit particle formation as well. Both She3myc and She1myc colocalized with the particle. Video microscopy demonstrated that She1p/Myo4p moved particles to the bud tip at 200-440 nm/sec. Therefore, the ASH1 3'UTR-dependent particle serves as a marker for RNA transport and localization.

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min −1 , much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.Transcription by RNA polymerase II (Pol II) is at the core of gene expression and hence is the basis of all cellular activities. Little information exists about the kinetics of this process in live cells 1 , as understanding of gene expression regulation comes from studies using purified proteins. For instance, the subunits of the elongating Pol II are well known 2 and the crystal structure of this enzyme explains much of its behavior in vitro 3,4 . mRNA transcription can be deconstructed into a succession of steps: promoter assembly, clearanceCorrespondence should be addressed to R.H.S. (rhsinger@aecom.yu.edu). AUTHOR CONTRIBUTIONS All data were initially acquired by X.D. and Y.S.-T. Subsequent data were obtained by V.d.T. (Fig. 4a,b and Fig. 5a) and Y.B. (Fig. 9b). S.M.S. was responsible for the microscopy, built the wide-field microscope for live-cell imaging and wrote analysis software. X.D. performed the kinetic modeling. R.D.P. provided consultation on model formulation and testing, and training in the use of the ProcessDB software. R.H.S. supervised the project. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/nsmb/. HHS Public AccessAuthor manuscript Nat Struct Mol Biol. Author manuscript; available in PMC 2016 July 12. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript and escape 5 , followed by elongation and termination. The process of transcriptional initiation involves several structural changes in the polymerase as the nascent transcript elongates 6 . Early in initiation, the polymerase can produce abortive transcripts 7,8 . These abortive cycles have been observed with a single prokaryote polymerase (RNAP) releasing several transcripts without escaping the promoter 9,10 . The elongation step can be regulated by pausing for various times, as demonstrated using prokaryotic polymerases in vitro 11,12 .For eukaryotic cells, attempts have been mad...

It has not been possible to view the transcriptional activity of a single gene within a living eukaryotic cell. It is therefore unclear how long and how frequently a gene is actively transcribed, how this is modulated during differentiation, and how transcriptional events are dynamically coordinated in cell populations. By means of an in vivo RNA detection technique , we have directly visualized transcription of an endogenous developmental gene. We found discrete "pulses" of gene activity that turn on and off at irregular intervals. Surprisingly, the length and height of these pulses were consistent throughout development. However, there was strong developmental variation in the proportion of cells recruited to the expressing pool. Cells were more likely to re-express than to initiate new expression, indicating that we directly observe a transcriptional memory. In addition, we used a clustering algorithm to reveal synchronous transcription initiation in neighboring cells. This study represents the first direct visualization of transcriptional pulsing in eukaryotes. Discontinuity of transcription may allow greater flexibility in the gene-expression decisions of a cell.

Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.