Making the message clear: visualizing mRNA localization.
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.
Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.Show MeSH
Related in: MedlinePlus
Mentions: More recently, amino-allyl labeling has been replaced by direct incorporation of Alexa Fluor-labeled nucleotides into the transcript in vitro.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 . 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.
Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.