Chapter 6
Visualization of Transiently Expressed mRNA in Plants
Using MS2
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
Abstract
RNA transport and localization are evolutionarily conserved processes that allow protein translation to
occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates,
embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules
to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through
plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA
transport to establish localized replication centers within infected cells and then to disseminate their
infectious genomes between cells and throughout the plant organism with the help of their movement
proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement
protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding
mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of
assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a
19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent proteintagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the
in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal
cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for
their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA
imaging data.
Key words RNA localization, RNA visualization, RNA transport, MS2 system, TMV, Movement
protein
1 Introduction
The life of an mRNA begins when its 5
0 region emerges from a
transcribed gene in the nucleus. The nascent pre-mRNA is sequentially associated with RNA-binding proteins (RBPs) that control
every aspect of its life, such as its maturation, its transport through
the nuclear pores and its localization to specific sites in the cytoplasm, its localized translation, and its degradation [1]. By determining the localization at which a protein is translated, RNA
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020
103
Visualization of Transiently Expressed mRNA in Plants
Using MS2
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
Abstract
RNA transport and localization are evolutionarily conserved processes that allow protein translation to
occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates,
embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules
to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through
plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA
transport to establish localized replication centers within infected cells and then to disseminate their
infectious genomes between cells and throughout the plant organism with the help of their movement
proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement
protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding
mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of
assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a
19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent proteintagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the
in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal
cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for
their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA
imaging data.
Key words RNA localization, RNA visualization, RNA transport, MS2 system, TMV, Movement
protein
1 Introduction
The life of an mRNA begins when its 5
0 region emerges from a
transcribed gene in the nucleus. The nascent pre-mRNA is sequentially associated with RNA-binding proteins (RBPs) that control
every aspect of its life, such as its maturation, its transport through
the nuclear pores and its localization to specific sites in the cytoplasm, its localized translation, and its degradation [1]. By determining the localization at which a protein is translated, RNA
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020
103