region) in mRNAs actively participate in mRNA maturation, localization, and translation by interacting with cellular proteins and
should not be modified. Moreover, retention of their activity
should be verified after introducing the RBP-binding MSL aptamers into the target mRNA sequence. The fluorescent signal intensity emitted by the tagged target mRNA is directly proportional to
the number of MSL aptamers added into the construct. Each MSL
aptamer is recognized and bound by one MCP dimer. While
24 MSL copies are sufficient to detect a single mRNA molecule
[58], this involves the incorporation of around 1.5 kb MSL RNA
into the RNA under investigation, which possibly affects mRNA
maturation and function. Moreover, given the repetitive identity of
the incorporated material, each MSL copy increases the risk of
recombination during cloning, which may impede the recovery of
a functional construct. Due to these reasons, it is advisable to
design different RNA constructs, each containing a specific number
of MSL repetitions, and to test the activity of the MSL repetitions
also at different positions within the construct, but avoiding insertion into any eventually identified zip code or regulatory element.
Once plasmid constructs are obtained, the expected size of the
mature transcript as well as normal mRNA accumulation kinetics
and retention of the encoded protein function should be verified. It
was recently shown that MCP molecules bound to their MSLs in
the tagged mRNA can significantly modify the turnover of shortlived mRNAs in Saccharomyces cerevisiae [59, 60]. This problem
was solved by modifying MCP-binding affinity and by enlarging the
linker sequence between each element of the MSL tandem
[61, 62], thus illustrating the effect which the MS2 system might
have on the target mRNAs. The second element of the system, the
MCP fused to a fluorescent protein (FP) reporter (MCP:FP), is
frequently targeted to the nucleus by incorporation of a nuclear
localization signal (NLS). This will decrease the cytoplasmic background and therefore increases the signal-to-noise ratio for the
labeled RNAs [50]. The amount of expressed MCP must be sufficiently high to ensure proper binding to the MSL array. While not
all MSL repeats are decorated with MCP [58], an overly increased
MCP expression level augments cytoplasmic background and
should be avoided. Therefore, rigorous controls must be conducted to verify that the amount of MCP:FP needed to visualize
the MSL-tagged RNA indeed shows fluorescence only in the presence of MSL-tagged RNA but not in the presence of MSL-free
control mRNA under the same conditions. In the absence of a
specific MSL-tagged RNA, the MCP:FP should not aggregate or
form other types of visible fluorescent particles that would strongly
compromise the specific RNA signal [63].
Adding MSLs to the TMV genome resulted in the loss of
infectivity. Thus, we decided to determine if the MP particles
formed also in transiently MP-expressing cells contain RNA.
RNA Imaging in Plants Using MS2
107
should not be modified. Moreover, retention of their activity
should be verified after introducing the RBP-binding MSL aptamers into the target mRNA sequence. The fluorescent signal intensity emitted by the tagged target mRNA is directly proportional to
the number of MSL aptamers added into the construct. Each MSL
aptamer is recognized and bound by one MCP dimer. While
24 MSL copies are sufficient to detect a single mRNA molecule
[58], this involves the incorporation of around 1.5 kb MSL RNA
into the RNA under investigation, which possibly affects mRNA
maturation and function. Moreover, given the repetitive identity of
the incorporated material, each MSL copy increases the risk of
recombination during cloning, which may impede the recovery of
a functional construct. Due to these reasons, it is advisable to
design different RNA constructs, each containing a specific number
of MSL repetitions, and to test the activity of the MSL repetitions
also at different positions within the construct, but avoiding insertion into any eventually identified zip code or regulatory element.
Once plasmid constructs are obtained, the expected size of the
mature transcript as well as normal mRNA accumulation kinetics
and retention of the encoded protein function should be verified. It
was recently shown that MCP molecules bound to their MSLs in
the tagged mRNA can significantly modify the turnover of shortlived mRNAs in Saccharomyces cerevisiae [59, 60]. This problem
was solved by modifying MCP-binding affinity and by enlarging the
linker sequence between each element of the MSL tandem
[61, 62], thus illustrating the effect which the MS2 system might
have on the target mRNAs. The second element of the system, the
MCP fused to a fluorescent protein (FP) reporter (MCP:FP), is
frequently targeted to the nucleus by incorporation of a nuclear
localization signal (NLS). This will decrease the cytoplasmic background and therefore increases the signal-to-noise ratio for the
labeled RNAs [50]. The amount of expressed MCP must be sufficiently high to ensure proper binding to the MSL array. While not
all MSL repeats are decorated with MCP [58], an overly increased
MCP expression level augments cytoplasmic background and
should be avoided. Therefore, rigorous controls must be conducted to verify that the amount of MCP:FP needed to visualize
the MSL-tagged RNA indeed shows fluorescence only in the presence of MSL-tagged RNA but not in the presence of MSL-free
control mRNA under the same conditions. In the absence of a
specific MSL-tagged RNA, the MCP:FP should not aggregate or
form other types of visible fluorescent particles that would strongly
compromise the specific RNA signal [63].
Adding MSLs to the TMV genome resulted in the loss of
infectivity. Thus, we decided to determine if the MP particles
formed also in transiently MP-expressing cells contain RNA.
RNA Imaging in Plants Using MS2
107