transcript abundance [14], grafting experiments show that mRNA
mobility is independent of the abundance of transcripts [5, 15]. Further studies show that in many phloem mobile mRNAs, the t-RNAlike motifs are necessary and sufficient to mediate RNA movement
[16]. Thus, it is possible that the movement of mobile mRNA is
controlled by a yet-to-be-elucidated regulatory mechanism. The
use of a mRNA live-imaging system provides an ideal approach to
reveal underlying regulatory elements.
One of the most frequently used approaches for genetics-based
mRNA live-cell imaging involves the use of the MS2 system
[17]. In this system, the coat protein of bacteriophage MS2,
which is an RNA-binding protein that specifically recognizes a
stem-loop (SL) structure in the bacteriophage RNA, is fused with
GFP and nuclear localization signal (NLS) from the SV40 virus.
When this MS2 SV40 -GFP fusion protein is co-expressed with target
mRNA conjugated to multiple copies of the SL structure, the
binding of MS2 SV40 -GFP to SL-bearing mRNAs reveals the location of the tagged mRNA by fluorescence. The NLS in the
MS2 SV40 -GFP fusion protein is used to restrict unbound
MS2 SV40 -GFP to the nucleus and, thus, to reduce the cytoplasmic
fluorescent background. The MS2 system has been widely used to
study cellular mRNA metabolism in various species, including
yeast, zebrafish, fly, mammals [18–21], and plants [22–25]. As
compared with the accumulated data for RNA live imaging in
yeast, mammals, and other systems, the data related to the MS2
system in plants are relatively limited [26]. The development of the
plant MS2 system is mainly hindered by the cytoplasmic background issue. The use of SV40 NLS allows a slight amount of
MS2 SV40 -GFP fusion protein to diffuse from the nucleus to cytosol
[27], thereby causing substantial fluorescent background in plant
cells. This drawback is significant in mature cells with extensive
vacuoles and restricted cytoplasmic space, such as leaf epidermal
cells, which are usually used for in vivo imaging.
Recently, we optimized the MS2 system for tracking mobile
mRNAs in planta [28]. Two improvements were achieved (1) by
replacement of the SV40 NLS (MS2 SV40 ) with the NLS from a
plant transcription factor FD (MS2 FD ) to diminish the cytoplasmic
fluorescent background and (2) by applying a specific sampling
strategy to optimize the appropriate time point for observation
(i.e., examining leaf cells at the boundary of the agrobacteriainfiltrated tissue patch that are close to the non-infiltrated surrounding tissues). Upon transient co-expression of MS2 FD -GFP
and SL-bearing target mRNAs in Nicotiana benthamiana cells, the
target mRNA is bound by MS2 FD -GFP and can be detected in the
cytosol by confocal microscopy. On the contrary, upon
co-expression of MS2 FD -GFP and target mRNAs without SL structures, the GFP fluorescence is exclusively detected in the nucleus.
Exceptional signal-to-noise ratio in cytosol indicates that the MS2
146
Kai-Ren Luo et al.
mobility is independent of the abundance of transcripts [5, 15]. Further studies show that in many phloem mobile mRNAs, the t-RNAlike motifs are necessary and sufficient to mediate RNA movement
[16]. Thus, it is possible that the movement of mobile mRNA is
controlled by a yet-to-be-elucidated regulatory mechanism. The
use of a mRNA live-imaging system provides an ideal approach to
reveal underlying regulatory elements.
One of the most frequently used approaches for genetics-based
mRNA live-cell imaging involves the use of the MS2 system
[17]. In this system, the coat protein of bacteriophage MS2,
which is an RNA-binding protein that specifically recognizes a
stem-loop (SL) structure in the bacteriophage RNA, is fused with
GFP and nuclear localization signal (NLS) from the SV40 virus.
When this MS2 SV40 -GFP fusion protein is co-expressed with target
mRNA conjugated to multiple copies of the SL structure, the
binding of MS2 SV40 -GFP to SL-bearing mRNAs reveals the location of the tagged mRNA by fluorescence. The NLS in the
MS2 SV40 -GFP fusion protein is used to restrict unbound
MS2 SV40 -GFP to the nucleus and, thus, to reduce the cytoplasmic
fluorescent background. The MS2 system has been widely used to
study cellular mRNA metabolism in various species, including
yeast, zebrafish, fly, mammals [18–21], and plants [22–25]. As
compared with the accumulated data for RNA live imaging in
yeast, mammals, and other systems, the data related to the MS2
system in plants are relatively limited [26]. The development of the
plant MS2 system is mainly hindered by the cytoplasmic background issue. The use of SV40 NLS allows a slight amount of
MS2 SV40 -GFP fusion protein to diffuse from the nucleus to cytosol
[27], thereby causing substantial fluorescent background in plant
cells. This drawback is significant in mature cells with extensive
vacuoles and restricted cytoplasmic space, such as leaf epidermal
cells, which are usually used for in vivo imaging.
Recently, we optimized the MS2 system for tracking mobile
mRNAs in planta [28]. Two improvements were achieved (1) by
replacement of the SV40 NLS (MS2 SV40 ) with the NLS from a
plant transcription factor FD (MS2 FD ) to diminish the cytoplasmic
fluorescent background and (2) by applying a specific sampling
strategy to optimize the appropriate time point for observation
(i.e., examining leaf cells at the boundary of the agrobacteriainfiltrated tissue patch that are close to the non-infiltrated surrounding tissues). Upon transient co-expression of MS2 FD -GFP
and SL-bearing target mRNAs in Nicotiana benthamiana cells, the
target mRNA is bound by MS2 FD -GFP and can be detected in the
cytosol by confocal microscopy. On the contrary, upon
co-expression of MS2 FD -GFP and target mRNAs without SL structures, the GFP fluorescence is exclusively detected in the nucleus.
Exceptional signal-to-noise ratio in cytosol indicates that the MS2
146
Kai-Ren Luo et al.