Making the message clear: visualizing mRNA localization.
Bottom Line: Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology.Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms.In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy.
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Mentions: More recently, amino-allyl labeling has been replaced by direct incorporation of Alexa Fluor-labeled nucleotides into the transcript in vitro.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.