Visualization of dynamics of single endogenous mRNA labeled in live mouse - PubMed (original) (raw)

Visualization of dynamics of single endogenous mRNA labeled in live mouse

Hye Yoon Park et al. Science. 2014.

Abstract

The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

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Figures

Fig. 1

Fig. 1

Labeled endogenous mRNA in MCP×MBS mouse. (A) Schematic for in vivo RNA labeling. NLS, nuclear localization sequence; HA, hemagglutinin; pol II, RNA polymerase II. (B and C) Single-particle tracking of GFP-labeled β-actin mRNP in primary MEF (B) and hippocampal neuron (C) from mouse. Track classifications: red, stationary; green, corralled; blue, diffusive; purple, directed motion (see supplementary text). N, nucleus; C, cytoplasm; S, soma. Scale bars, 10 μm. (D and E) Intensity histograms of β-actin mRNPs tracked in primary MEFs (D) and neurons (E). Red curves show one- and three-component Gaussian fits for (D) and (E), respectively. (F and G) Fraction of stationary, corralled, diffusive, and directed β-actin mRNPs in primary MEFs (F) and neurons (G). Error bars denote SEM (n = 6 cells).

Fig. 2

Fig. 2

Activity-dependent dynamics of β-actin mRNP in neurons from MCP×MBS mouse. (A and B) Examples of β-actin mRNP merge (A) and split (B) events. From top to bottom: initial images of the particles, kymographs during a 12-s period, and final images. (C) Ratio of split to merge events increased after KCl depolarization (n = 11 neurons from three cultures, 14 to 16 days in vitro; P < 0.05 by t test). (D) CA1 region in acute hippocampal slice. Scale bar, 50 μm. (E) Soma layer of CA1 neurons. Panels show maximum projections of x-y, y-z, and x-z planes. Scale bar, 10 μm. (F) Transcription of β-actin gene with KCl depolarization. Arrowheads denote β-actin transcription sites. Scale bar, 5 μm. (G) Nascent mRNA per transcription site increased after depolarization (n = 86 sites, brain slices from 3 mice; P < 0.001 by pairwise t test). Error bars denote SEM.

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References

    1. Tyagi S. Nat Methods. 2009;6:331–338. - PubMed
    1. Bertrand E, et al. Mol Cell. 1998;2:437–445. - PubMed
    1. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Science. 2002;295:868–872. - PubMed
    1. Lionnet T, et al. Nat Methods. 2011;8:165–170. - PMC - PubMed
    1. Bunnell TM, Burbach BJ, Shimizu Y, Ervasti JM. Mol Biol Cell. 2011;22:4047–4058. - PMC - PubMed

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