Live Imaging of Endogenous RNA Reveals a Diffusion and Entrapment Mechanism for nanos mRNA Localization in Drosophila

Forrest, Kevin M.; Gavis, Elizabeth R.

doi:10.1016/s0960-9822(03)00451-2

Cited by 383 publications

(395 citation statements)

References 55 publications

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“…When an MS2-GFP fusion is coexpressed with a reporter RNA containing tandemly repeated MS2-binding sites, the fusion protein binds to the RNA, forming bright fluorescent particles. This method was first used in vivo to follow mRNA localization in yeast (19) and Drosophila (21) and was used to detect single mRNA molecules in mammalian cells (20). We found, not surprisingly, that several modifications of this system were required for use in E. coli.…”

Section: Methodsmentioning

confidence: 98%

“…Our approach is based on a method developed by Singer and coworkers (19,20), which has been used to track RNA in various eukaryotic systems (21). The mRNA detection system is comprised of two elements, a fluorescence protein fused to the RNA bacteriophage MS2 coat protein (henceforth referred to as MS2) and a reporter RNA containing tandemly repeated MS2-binding sites.…”

Section: Methodsmentioning

confidence: 99%

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RNA dynamics in live Escherichia coli cells

Golding

Cox

2004

Proc. Natl. Acad. Sci. U.S.A.

276

290

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We describe a method for tracking RNA molecules in Escherichia coli that is sensitive to single copies of mRNA, and, using the method, we find that individual molecules can be followed for many hours in living cells. We observe distinct characteristic dynamics of RNA molecules, all consistent with the known life history of RNA in prokaryotes: localized motion consistent with the Brownian motion of an RNA polymer tethered to its template DNA, free diffusion, and a few examples of polymer chain dynamics that appear to be a combination of chain fluctuation and chain elongation attributable to RNA transcription. We also quantify some of the dynamics, such as width of the displacement distribution, diffusion coefficient, chain elongation rate, and distribution of molecule numbers, and compare them with known biophysical parameters of the E. coli system.T he bacterium Escherichia coli may well be the most thoroughly characterized model biological system. Much of its metabolism serves as the basic paradigm for DNA replication, RNA transcription, protein synthesis, and gene regulation (1, 2).Recently, techniques have become available that allow us to study central problems in genome organization and expression in individual living cells (3, 4), rather than rely on the averaged properties of large populations. These studies have added to our knowledge in two main areas: the heterogeneity among cells in a supposedly homogeneous population (5-7) and the spatiotemporal organization of macromolecules in the bacterial cell (8). The subcellular location of a variety of different proteins involved in cell division, such as the MinCDE family (9), are now known to be localized in special regions, as is the location of replicating chromosomes (10) and several different plasmids (11,12). Complexes of signaling proteins in Bacillus subtilis and Caulobacter crescentus have likewise been localized to the poles of dividing and differentiating cells (8, 13).To our knowledge, there are no published studies on the localization and timing of mRNA synthesis in living bacterial cells. Our knowledge of RNA transcription and dynamics comes from population studies, or in vitro studies with purified components (14-17). A rare glimpse into cell-to-cell variability in message number was provided by the studies of with Salmonella using whole fixed mounts. However, there is no information at the single-cell level in living cells on message localization: whether mRNA is free to move throughout the cytoplasm, whether movement is active or passive, and, for RNA molecules present in low copy numbers, how the actual number of molecules is a function of cell state. This last information is of particular interest because it bears directly on the question of the regulation of proteins known to be present in low copy numbers (for example, many of the proteins that act to switch genes on and off) and how the cell successfully regulates copy number in the presence of considerable extrinsic and intrinsic noise (see ref. 7 for a review).Here we describe a metho...

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Section: Methodsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

RNA dynamics in live Escherichia coli cells

Golding

Cox

2004

Proc. Natl. Acad. Sci. U.S.A.

276

290

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“…Genetic and molecular data support a model where localization activates translation by preventing repressors from interacting with the RNA (23,67). Live cell imaging shows nanos RNA in particles, from 0.1 to 1 mm, but only upon localization at the posterior pole, further suggesting specific proteins can only assemble with it at this location (48).…”

Section: Translational Controlmentioning

confidence: 88%

“…Importantly, anchoring does not seem to depend on a specialized posterior cortex, but rather the polarized microtubule based transport of osk RNA to the posterior (59). Actin cytoskeleton is also required for the anchoring of the germ plasm to the subcortex and its disruption results in intact germ plasm being lost from the subcortical region (48).…”

Section: Cortical Anchoring Of Localized Rnas: the Cytoskeletonmentioning

confidence: 99%

Sending RNAs into the Future: RNA Localization and Germ Cell Fate

King

2004

IUBMB Life

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RNA localization is a cellular mechanism used to localize proteins to subcellular domains and to control protein synthesis regionally. In oocytes, RNA localization has profound implications for development, setting up local concentrations of regulatory proteins that will establish regional fates in the future embryo. One such fate is that of the germ cell lineage. In a diverse number of organisms, including Drosophila and Xenopus, the germ cell lineage is determined by the inheritance of germ plasm assembled during oogenesis. This process requires the recruitment of specific RNAs, many now identified, to the germ plasm. Complex signals located in the 3' UTR direct RNAs to their destinations. These signals are sites for protein binding and assembly into particles competent to localize. Three different mechanisms have been described that operate during oogenesis or embryogenesis to localize RNAs in the germ plasm: motor driven transport, differential stability, and entrapment. Each of these localization mechanisms must be coordinated with translation and anchoring mechanisms to achieve functional germ plasms. Here we review what is known about these processes in germ cells, but the cellular mechanisms that select and transport RNAs are likely to be conserved among somatic cells as well. IUBMB Life, 56: 19‐27, 2004

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“…This approach has been successfully applied to visualize the behavior of several mRNAs in different organisms. [18][19][20] We recently adopted it to produce a version of TLC1 bearing ten MS2 stem loops near the 3' end of the mature RNA. 16 Co-expression of this tagged TLC1-10xMS2 RNA with the MS2 coat protein fused to GFP yielded in several fluorescent foci in yeast nuclei (named hereafter TLC1-GFP).…”

Section: Dynamics In Yeastmentioning

confidence: 99%

Telomerase caught in the act

et al. 2012

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T he stable linearity of eukaryotic chromosomes depends on special characteristics of their ends, the telomeres. Accurate telomere function in turn requires a sustained presence of repeated DNA elements, which are maintained by the enzyme telomerase. The telomerase holoenzyme is composed of both protein and RNA, and its functions rely on proper expression, maturation, trafficking and assembly of these components. Conflicting models for the recruitment of telomerase at telomeres have been proposed; one suggests a local activation of telomerase at short telomeres, while the other proposes that telomerase is recruited only at short telomeres. To discriminate between these models and investigate the cell cycle-dependent regulation of telomerase in living cells, a GFP reporter system to visualize the yeast telomerase RNA has been recently developed. This assay shed new light on the mechanism of recruitment of telomerase to telomeres, and it uncovered a hitherto unrecognized mechanism for restricting telomerase access to telomeres.

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Live Imaging of Endogenous RNA Reveals a Diffusion and Entrapment Mechanism for nanos mRNA Localization in Drosophila

Abstract: These results reveal a diffusion-based, late-acting posterior localization mechanism for long-range transport of nanos mRNA. This mechanism differs from directed transport-based localization mechanisms in its reliance on bulk movement of RNA.

Cited by 383 publications

References 55 publications

RNA dynamics in live Escherichia coli cells

RNA dynamics in live Escherichia coli cells

Sending RNAs into the Future: RNA Localization and Germ Cell Fate

Telomerase caught in the act

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