bimolecular fluorescence complementation between two RBP fusion
proteins bound to the same RNA. Some RBPs used for RNA imaging can be engineered to bind to a sequence of choice, enabling
detection of native, unaltered RNAs [4]. However, the effects of
artificially altered sequence specificity on RNA affinity can be hard to
predict [10]. Alternatively, the RNA of interest can be genetically
tagged with a sequence motif bound by a suitable RBP. This allows
for the use of well-characterized protein-RNA interactions, and
sensitivity can easily be increased by using multiple tandem copies
of the recognition motif. The latter approach has been implemented
with several stem-loop-binding RBPs, first with the capsid protein of
bacteriophage MS2 and later with several similar systems [8, 9 and
references therein]. The dissociation constants of the respective
protein-RNA complexes vary between about 1 nM and 3 μM. In
order to achieve sensitive RNA imaging, typically 6–24 stem-loops
are inserted into the RNA of interest, although as few as 4 and as
many as 96 have been employed [8, 9 and references therein]. The
extensive secondary structures formed by these tags can interfere
with RNA processing, localization, and translation [11, 12]. For
imaging of viral genomes, multiple tandem stem-loops are particularly problematic, as RNA viruses rely on native secondary structures
and short- and long-range intramolecular base pairing for regulation
of their RNA replication and gene expression.
Here, we describe RNA tagging for imaging with the bacterial
endonuclease Csy4 (21.4 kDa), which recognizes its cognate,
15 bp stem-loop (Fig. 1a) with exceptionally high affinity (k D
0.05 nM) [13]. Csy4 normally functions in the processing of
CRISPR pre-crRNAs, which it cleaves specifically immediately
after the recognized stem-loop. This endonuclease activity can be
removed with a single-point mutation in the active site, H29A,
which does not affect RNA affinity [14, 16]. By fusing Csy4
[H29A] (Csy4∗) to GFP and tagging RNAs of interest with two
cognate stem-loops, highly sensitive imaging of Potato virus X
(PVX) vRNA was achieved in infected Nicotiana benthamiana
leaf epidermal cells (Burnett & Tilsner, in preparation), while the
distribution of messenger RNAs tagged with 12 stem-loops was
visualized in hyphae of the filamentous fungus Neurospora crassa
(Tilsner & Lichius, in preparation).
2 Materials
Novel and published plasmids described in this chapter are available
upon request for noncommercial academic research.
2.1 Construction of
Csy4∗ Expression
Plasmids for Imaging
in Plants
1. Synthesized gene fragment of Pseudomonas aeruginosa
UCBPP-PA14 Csy4∗ open reading frame (https://www.
ncbi.nlm.nih.gov/nuccore/NC_008463.1?from¼2927517&
to¼2928080) without the stop codon (see Note 1). Following
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David Burnett et al.
proteins bound to the same RNA. Some RBPs used for RNA imaging can be engineered to bind to a sequence of choice, enabling
detection of native, unaltered RNAs [4]. However, the effects of
artificially altered sequence specificity on RNA affinity can be hard to
predict [10]. Alternatively, the RNA of interest can be genetically
tagged with a sequence motif bound by a suitable RBP. This allows
for the use of well-characterized protein-RNA interactions, and
sensitivity can easily be increased by using multiple tandem copies
of the recognition motif. The latter approach has been implemented
with several stem-loop-binding RBPs, first with the capsid protein of
bacteriophage MS2 and later with several similar systems [8, 9 and
references therein]. The dissociation constants of the respective
protein-RNA complexes vary between about 1 nM and 3 μM. In
order to achieve sensitive RNA imaging, typically 6–24 stem-loops
are inserted into the RNA of interest, although as few as 4 and as
many as 96 have been employed [8, 9 and references therein]. The
extensive secondary structures formed by these tags can interfere
with RNA processing, localization, and translation [11, 12]. For
imaging of viral genomes, multiple tandem stem-loops are particularly problematic, as RNA viruses rely on native secondary structures
and short- and long-range intramolecular base pairing for regulation
of their RNA replication and gene expression.
Here, we describe RNA tagging for imaging with the bacterial
endonuclease Csy4 (21.4 kDa), which recognizes its cognate,
15 bp stem-loop (Fig. 1a) with exceptionally high affinity (k D
0.05 nM) [13]. Csy4 normally functions in the processing of
CRISPR pre-crRNAs, which it cleaves specifically immediately
after the recognized stem-loop. This endonuclease activity can be
removed with a single-point mutation in the active site, H29A,
which does not affect RNA affinity [14, 16]. By fusing Csy4
[H29A] (Csy4∗) to GFP and tagging RNAs of interest with two
cognate stem-loops, highly sensitive imaging of Potato virus X
(PVX) vRNA was achieved in infected Nicotiana benthamiana
leaf epidermal cells (Burnett & Tilsner, in preparation), while the
distribution of messenger RNAs tagged with 12 stem-loops was
visualized in hyphae of the filamentous fungus Neurospora crassa
(Tilsner & Lichius, in preparation).
2 Materials
Novel and published plasmids described in this chapter are available
upon request for noncommercial academic research.
2.1 Construction of
Csy4∗ Expression
Plasmids for Imaging
in Plants
1. Synthesized gene fragment of Pseudomonas aeruginosa
UCBPP-PA14 Csy4∗ open reading frame (https://www.
ncbi.nlm.nih.gov/nuccore/NC_008463.1?from¼2927517&
to¼2928080) without the stop codon (see Note 1). Following
158
David Burnett et al.