Chapter 9
RNA Imaging with RNase-Inactivated Csy4 in Plants
and Filamentous Fungi
David Burnett, Alexander Lichius, and Jens Tilsner
Abstract
Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a
sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems
have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for
imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity
for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few
tandem copies of the target stem-loop inserted into the RNA of interest.
Key words Csy4, RNA stem-loop, RNA imaging, Live-cell imaging, RNA virus, Plant virus, Filamentous fungi, Neurospora crassa
1 Introduction
The subcellular localizations of various types of RNA are intimately
related to their functions. Functional localizations can include
highly localized translation or recruitment to various types of
RNA granules [1–3]. Viral RNA (vRNA) genomes are localized at
sites of replication, translation, and encapsidation, and in plants
some types of RNA are also transported between cells [4, 5]. In
filamentous fungi, targeted delivery of RNAs provides essential
means of spatial control over their respective function within the
elaborate hyphal network [6, 7].
Multiple systems have been developed that enable sequencespecific tracking of RNA molecules in living cells [8, 9]. Genetically
encoded RNA reporters are particularly well suited for RNA imaging
in organisms with a cell wall, as they do not necessitate delivery of
additional molecules into cells. Generally, they consist of a sequencespecific RNA-binding protein (RBP) translationally fused to a fluorescent protein. Unbound and RNA-bound reporter can be distinguished either by directing the unbound fusion protein to the
nucleus, from where it is recruited by cytoplasmic RNAs, or by
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020
157
RNA Imaging with RNase-Inactivated Csy4 in Plants
and Filamentous Fungi
David Burnett, Alexander Lichius, and Jens Tilsner
Abstract
Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a
sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems
have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for
imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity
for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few
tandem copies of the target stem-loop inserted into the RNA of interest.
Key words Csy4, RNA stem-loop, RNA imaging, Live-cell imaging, RNA virus, Plant virus, Filamentous fungi, Neurospora crassa
1 Introduction
The subcellular localizations of various types of RNA are intimately
related to their functions. Functional localizations can include
highly localized translation or recruitment to various types of
RNA granules [1–3]. Viral RNA (vRNA) genomes are localized at
sites of replication, translation, and encapsidation, and in plants
some types of RNA are also transported between cells [4, 5]. In
filamentous fungi, targeted delivery of RNAs provides essential
means of spatial control over their respective function within the
elaborate hyphal network [6, 7].
Multiple systems have been developed that enable sequencespecific tracking of RNA molecules in living cells [8, 9]. Genetically
encoded RNA reporters are particularly well suited for RNA imaging
in organisms with a cell wall, as they do not necessitate delivery of
additional molecules into cells. Generally, they consist of a sequencespecific RNA-binding protein (RBP) translationally fused to a fluorescent protein. Unbound and RNA-bound reporter can be distinguished either by directing the unbound fusion protein to the
nucleus, from where it is recruited by cytoplasmic RNAs, or by
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020
157