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Making the message clear: visualizing mRNA localization.

Weil TT, Parton RM, Davis I - Trends Cell Biol. (2010)

Bottom Line: Most significantly, methods for following the movement of mRNA in living cells are now in widespread use.These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components.These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

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Detection of RNA in live tissue. (a) Localization of multiple RNAs via injection. Drosophila syncytial blastoderm expressing nuclear GFP (blue) first injected with an Alexa Fluor 546-labeled runt RNA (red) and shortly after with an Alexa Fluor 488-labeled runt RNA (green). Both RNAs were injected into the same site (arrow). The image shows the first RNA is already apically localized, whereas the second is in transit (courtesy of Renald Delanoue). (b) Tracking individual β-actin mRNA molecules in vivo with MTRIPs in a human epithelial cell. Images were taken at 0.5 Hz for 5 min. Areas of interest (b′–b′″) show trajectories of single mRNA granules (courtesy of Philip Santangelo) [34].
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fig3: Detection of RNA in live tissue. (a) Localization of multiple RNAs via injection. Drosophila syncytial blastoderm expressing nuclear GFP (blue) first injected with an Alexa Fluor 546-labeled runt RNA (red) and shortly after with an Alexa Fluor 488-labeled runt RNA (green). Both RNAs were injected into the same site (arrow). The image shows the first RNA is already apically localized, whereas the second is in transit (courtesy of Renald Delanoue). (b) Tracking individual β-actin mRNA molecules in vivo with MTRIPs in a human epithelial cell. Images were taken at 0.5 Hz for 5 min. Areas of interest (b′–b′″) show trajectories of single mRNA granules (courtesy of Philip Santangelo) [34].

Mentions: More recently, amino-allyl labeling has been replaced by direct incorporation of Alexa Fluor-labeled nucleotides into the transcript in vitro. Injecting RNA labeled in this way into live cells led to the formation of bright RNP particles (Figure 3a), and analysis of their movements revealed that transport occurs along microtubules by the molecular motor dynein and enabled one of the earliest determinations of RNA transport particle speeds [30]. This assay has also facilitated the characterization of cis-acting elements within the RNA sequence and the requirement for trans-acting factors [31,32]. Whereas in vitro RNA synthesis is very rapid and does not require lengthy transgenic experiments, the microinjection procedure is potentially damaging, and introducing a large excess of RNA can saturate intracellular machinery leading to inappropriate localization and expression.


Making the message clear: visualizing mRNA localization.

Weil TT, Parton RM, Davis I - Trends Cell Biol. (2010)

Detection of RNA in live tissue. (a) Localization of multiple RNAs via injection. Drosophila syncytial blastoderm expressing nuclear GFP (blue) first injected with an Alexa Fluor 546-labeled runt RNA (red) and shortly after with an Alexa Fluor 488-labeled runt RNA (green). Both RNAs were injected into the same site (arrow). The image shows the first RNA is already apically localized, whereas the second is in transit (courtesy of Renald Delanoue). (b) Tracking individual β-actin mRNA molecules in vivo with MTRIPs in a human epithelial cell. Images were taken at 0.5 Hz for 5 min. Areas of interest (b′–b′″) show trajectories of single mRNA granules (courtesy of Philip Santangelo) [34].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902723&req=5

fig3: Detection of RNA in live tissue. (a) Localization of multiple RNAs via injection. Drosophila syncytial blastoderm expressing nuclear GFP (blue) first injected with an Alexa Fluor 546-labeled runt RNA (red) and shortly after with an Alexa Fluor 488-labeled runt RNA (green). Both RNAs were injected into the same site (arrow). The image shows the first RNA is already apically localized, whereas the second is in transit (courtesy of Renald Delanoue). (b) Tracking individual β-actin mRNA molecules in vivo with MTRIPs in a human epithelial cell. Images were taken at 0.5 Hz for 5 min. Areas of interest (b′–b′″) show trajectories of single mRNA granules (courtesy of Philip Santangelo) [34].
Mentions: More recently, amino-allyl labeling has been replaced by direct incorporation of Alexa Fluor-labeled nucleotides into the transcript in vitro. Injecting RNA labeled in this way into live cells led to the formation of bright RNP particles (Figure 3a), and analysis of their movements revealed that transport occurs along microtubules by the molecular motor dynein and enabled one of the earliest determinations of RNA transport particle speeds [30]. This assay has also facilitated the characterization of cis-acting elements within the RNA sequence and the requirement for trans-acting factors [31,32]. Whereas in vitro RNA synthesis is very rapid and does not require lengthy transgenic experiments, the microinjection procedure is potentially damaging, and introducing a large excess of RNA can saturate intracellular machinery leading to inappropriate localization and expression.

Bottom Line: Most significantly, methods for following the movement of mRNA in living cells are now in widespread use.These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components.These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Show MeSH
Related in: MedlinePlus