RNA Detection

Chapter 21

RNA Live Imaging in the Model MicroorganismUstilago

maydis

Sabrina Zander, Kira M€untjes, and Michael Feldbr€ugge

Abstract

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 3^0 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 microorganismUstilago maydis. The presented experimental
design and the microscopic techniques are applicable to a broad range of other organisms.

Key wordsmRNPs, Dual color microscopy, Endosomal mRNA transport, Early endosomes, Micro-

tubule-dependent transport

1 Introduction

Every cell needs to determine precisely when and where mRNAs are

to be translated into proteins. This spatiotemporal expression is, for

example, crucial to support compartmentalization in eukaryotic

cells. Therefore, it is rather common that mRNAs localize to a

specific subcellular site in the cytoplasm for the precise delivery of

the encoded protein [1–4]. A prominent mechanism for mRNA

localization is their active transport along the cytoskeleton [5–7].

For long-distance movement mRNAs are transported along micro-

tubules [5]. A well-studied example in eukaryotic microorganisms

is endosomal mRNP transport inU. maydis, a trafficking process

that is needed for the efficient unipolar growth of infectious hyphae

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_21,©Springer Science+Business Media LLC 2018

Sabrina Zander and Kira Muntjes contributed equally to this work.€

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