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Visualizing RNA movement in real time in living cells

    Researchers have been forced for many years to interpret cellular data based on fixed cells.  Often the progression of particles in the cell had to be synthesized using multiple cells fixed at different times in hopes that the cells would sketch a pattern of particle motility.
    Recently, a novel method has been developed to visualize RNA in living cells  (Bertrand and others 1998).  Yeast cells are cotransformed using two plasmids, a lacZ reporter mRNA and a GFP-MS2 fusion protein.  The reporter mRNA usually contains six MS2 binding sites, each with a 19 nucleotide stem-loop, inserted directly after the lacZ encoding sequence termination.  The multiple stem-loop localization determinants in the insertion allow for multiple green fluorescent protein (GFP) binding sites, thus increasing the fluorescence of the cell (Chartrand and others 1999).  The other plasmid contained a GFP sequence fused to coding sequences for MS2, a single-stranded RNA bacteriophage capsid protein (Figure 1A).  The mRNA reporters are expressed under the influence of GAL promoters, a galactose-inducible promoter, while the fusion proteins are controlled by the constitutive GPD promoter.

Figure 1. A (top) Schematic of the MS2-GFP Chimeric Protein.  The protein is controlled by GPD promoter which is followed by a nuclear localization signal (NLS) and HA tag at the N terminus to insure only target bound GFP protein will by in the cytoplasm.  Following the HA tag is the MS2-GFP protein and the protein terminator.  (bottom) Schematic of mRNA reporters controlled by GAL promoters.  The reporters contain a 5' intron followed by the lacZ encoding sequence and six MS2 binding sites immediately after the translation termination codon.  The 3' untranslated region of the ASH1 gene, which contains the localization signal in yeast, follows the MS2 binding sites.
B.  Live cells expressing the lacZ-MS2-ASH1 reporter mRNA and GFP-MS2 fusion protein.
(See Bertrand et al., Molecular Cell 2:437-445 (1998))

  The GFP-lac Z-MS2 reporter mRNA produces a bright fluorescent particle, RNA, which can be localized within the cell (Chartrand, Meng, Singer, Long 1999).  The particles are bright enough to follow in living cells using real time digital imaging and video microscopy, allowing for analysis of motion, speed, and location of particles (Beach DL, Salmon ED, Bloom K 1999; Bertrand and others 1998).  High levels of background fluorescence is inhibited by the addition of a nuclear localization signal and HA tag, which allow only GFP-MS2 chimera to reside in the cytoplasm.  Now excluding any unbound GFP, the quantity of reporter mRNA at a particular location can be determined by monitoring the accumulation of fluorescent protein synthesized at the target area (Long and others 1997).  Using a video camera connected to a VCR, moving particles within live cells mounted between two cover slips can be identified by their fluorescence and followed for up to four minutes.  Live-cell-time-lapse images made with a digital camera can reveal movements dependent on cellular cycles within the same cell (Bertrand and others 1998).  The cells can be constructed using a digital camera and 3D Reconstruction software which allows multiple point views meshed together to form a congruent image (Beach DL, Salmon ED, Bloom K 1999).

Figure 2.  Analysis of particle movement.  Bright field microscopy and epifluorescence were used to observe wild-type yeast expressing both GFP-protein and ASH1 reporter.  Particle movement was observed using a video camera linked to a VCR.  (Time progression is represented by progression from dark to bright colors.)  (See Bertrand et al., Molecular Cell 2:437-445 (1998))

To ensure correct attribution of bright particles to the GFP-MS2/reporter mRNA complex, fluorescent in situ hybridization (FISH) can be performed using probes for lacZ or MS2.  As detection of RNA is more sensitive using FISH, fluorescent particles corresponding to one another in both procedures indicates a complexed GFP-MS2/reporter mRNA (Bertrand and others 1998).  RNA processing and transport is too fast for FISH to detect subtleties in position and localization, therefore FISH is better equipped to confirm results rather than detect them.

Figure 3. Localization of ASH1 mRNA and Ash1p to daughter cells of budding yeast using FISH.
(See Long et al., Science 277:383-386 (1997))

Although single RNA molecules cannot be visualized using GFP, investigation of any RNA-protein complex, such as RNA processing, nuclear export, or intracellular targeting can be performed via in vivo localization.  The method is applicable not only to yeast cells as presented but also to higher eukaryotic cells (Bertrand and others 1998).

To learn more about visualizing RNA movement in living cells check out these sites:


Beach DL, Salmon ED, Bloom K.  1999.  Localization and anchoring of mRNA in budding yeast.
    Current Biology  9:569-578.
Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM.  1998.  Localization of
    ASH1 mRNA particles in living yeast.  Molecular Cell 2:437-445.
Chartrand P, Meng X, Singer RH, Long RM.  1999.  Structural elements required for the
    localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo.  Current
    Biology  9:333-336.
Long RM, Singer RH, Meng X, Gonzalez I, Nasmyth K, Jansen RP.  1997.  Mating type switching
    in yeast controlled by asymmetric localization of ASH1 mRNA.  Science  277:383-386.

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