However, fixed cells provide limited information about highly
dynamic and rare events controlling mRNA metabolism. To follow
mRNAs in living cells, several labeling strategies have been developed over the past decades. The best characterized system is a
genetically encoded reporter based on the multimerization of
RNA stem-loops derived from the bacteriophage MS2 [40–
42]. To visualize single mRNAs in living cells, 24 MS2 stemloops are used to tag an RNA of interest, which is then detected
by co-expression of a specific RNA-binding protein, the MS2 coat
protein (MCP), fused to fluorescent proteins (FP, i.e., eGFP,
mCherry, tdTomato, HALO, photoactivatable proteins)
[1, 39]. Several MS2 array variants are available, and the recommendation for using a particular one depends on the model organism, the mRNA, and the step of the mRNA life cycle under
investigation [1, 43]. High-affinity MS2-MCP variants have been
successfully used to measure the dynamics of mRNA transcription
and splicing [44–46], export [47, 48], localization [49, 50], and
translation in mammalian cells [51–55]. They are also recommended if the experimental setup involves FRAP. However, several
groups reported that the high-affinity MS2-MCP variants are not
optimal to visualize mRNAs in rapidly dividing organisms such as
S. cerevisiae [56–59]. For this reason, we recently generated an
improved MS2 array (MS2-binding sites V6, MBSV6) with
decreased affinity for MCP. This allowed measuring the half-life of
rapidly decaying mRNAs while preserving single-molecule mRNA
detection in living cells [43, 58]. Low-affinity MS2-MCP variants
have also been used to tag mRNAs in mammalian cells, specifically
to generate arrays containing up to 128 MS2 stem-loops in a single
transcript, with the aim of monitoring transcription with high
temporal resolution for long periods of time and with minimal
photo-bleaching [60].
To visualize more than one mRNA species at the time, in single
living cells, several orthogonal systems are available. Another system was generated by multimerizing RNA stem-loops derived from
the bacteriophage PP7, detected by the cognate protein PP7 coat
protein (PCP) fused to fluorescent proteins [61]. This reporter has
been used to study transcription dynamics [62], mRNA export
[63], and translation [64, 65]. It has also been used to create a
homozygous mice where the immediate early gene Arc was endogenously tagged with 24 PP7 loops, allowing to visualize its response
to synaptic activity [66]. Alternative genetically encoded RNA
labeling strategies use arrays generated from other RNA sequences,
such as the BglG stem-loop [67], the λ BoxB RNA [68], and the
U1A loop [69, 70]. Other RNA labeling methods are reviewed
elsewhere [1, 43, 54].
The optimization of single mRNA visualization in living cells
relies also on the expansion of the FP and fluorophore palette
[71, 72]. In a recent publication, the brightness, photostability,
pH resistance, and monomeric properties of more than 40 FP have
Imaging Single mRNAs in Living Eukaryotic Cells
123
dynamic and rare events controlling mRNA metabolism. To follow
mRNAs in living cells, several labeling strategies have been developed over the past decades. The best characterized system is a
genetically encoded reporter based on the multimerization of
RNA stem-loops derived from the bacteriophage MS2 [40–
42]. To visualize single mRNAs in living cells, 24 MS2 stemloops are used to tag an RNA of interest, which is then detected
by co-expression of a specific RNA-binding protein, the MS2 coat
protein (MCP), fused to fluorescent proteins (FP, i.e., eGFP,
mCherry, tdTomato, HALO, photoactivatable proteins)
[1, 39]. Several MS2 array variants are available, and the recommendation for using a particular one depends on the model organism, the mRNA, and the step of the mRNA life cycle under
investigation [1, 43]. High-affinity MS2-MCP variants have been
successfully used to measure the dynamics of mRNA transcription
and splicing [44–46], export [47, 48], localization [49, 50], and
translation in mammalian cells [51–55]. They are also recommended if the experimental setup involves FRAP. However, several
groups reported that the high-affinity MS2-MCP variants are not
optimal to visualize mRNAs in rapidly dividing organisms such as
S. cerevisiae [56–59]. For this reason, we recently generated an
improved MS2 array (MS2-binding sites V6, MBSV6) with
decreased affinity for MCP. This allowed measuring the half-life of
rapidly decaying mRNAs while preserving single-molecule mRNA
detection in living cells [43, 58]. Low-affinity MS2-MCP variants
have also been used to tag mRNAs in mammalian cells, specifically
to generate arrays containing up to 128 MS2 stem-loops in a single
transcript, with the aim of monitoring transcription with high
temporal resolution for long periods of time and with minimal
photo-bleaching [60].
To visualize more than one mRNA species at the time, in single
living cells, several orthogonal systems are available. Another system was generated by multimerizing RNA stem-loops derived from
the bacteriophage PP7, detected by the cognate protein PP7 coat
protein (PCP) fused to fluorescent proteins [61]. This reporter has
been used to study transcription dynamics [62], mRNA export
[63], and translation [64, 65]. It has also been used to create a
homozygous mice where the immediate early gene Arc was endogenously tagged with 24 PP7 loops, allowing to visualize its response
to synaptic activity [66]. Alternative genetically encoded RNA
labeling strategies use arrays generated from other RNA sequences,
such as the BglG stem-loop [67], the λ BoxB RNA [68], and the
U1A loop [69, 70]. Other RNA labeling methods are reviewed
elsewhere [1, 43, 54].
The optimization of single mRNA visualization in living cells
relies also on the expansion of the FP and fluorophore palette
[71, 72]. In a recent publication, the brightness, photostability,
pH resistance, and monomeric properties of more than 40 FP have
Imaging Single mRNAs in Living Eukaryotic Cells
123