An essential feature of protein expression is the tight regulation of when and where a protein is translated from its cognate mRNA. This spatiotemporal expression is particularly important in guaranteeing the correct and efficient targeting of proteins to defined subcellular sites. In order to achieve local translation, mRNAs must be deposited at specific locations. A common mechanism is the active transport of mRNAs along the actin or microtubule cytoskeleton. To study such dynamic transport processes in vivo RNA live imaging is the method of choice. This method is based on the principle that defined binding sites for a heterologous RNA-binding protein (RBP) are inserted in the 3' UTR of target mRNAs. Coexpression of the RBP fused to a fluorescent protein enables mRNA detection in vivo using fluorescence microscopy techniques. In this chapter we describe the well-established method of studying microtubule-dependent mRNA transport in the eukaryotic model microorganism Ustilago maydis. The presented experimental design and the microscopic techniques are applicable to a broad range of other organisms.
Keywords: Dual color microscopy; Early endosomes; Endosomal mRNA transport; Microtubule-dependent transport; mRNPs.