NEWS AND VIEWS
Splitting or stacking fluorescent
proteins to visualize mRNA in
living cells
Sanjay Tyagi
Several new approaches to tag mRNA allow real-time imaging of mRNA
dynamics in living bacteria, yeast and human cells.
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Kim Caesar
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As every mRNA molecule yields many protein molecules upon translation, by localizing
mRNAs within specific subcellular territories,
cells can exert powerful control over where
and when a protein is expressed. In fact, subcellular localization of mRNA has a key role
in processes as diverse as morphogenesis, cell
migration and memory formation. To dissect
the processes that lead to intracellular mRNA
localization and to study other RNA dynamics, cell biologists have long sought to image
mRNAs in the natural context of living cells.
So far, most efforts have focused on developing nucleic-acid probes that become fluorescent upon binding to a region of an mRNA4
or on tagging RNAs with motifs that bind to
fluorescent proteins5. The latter approach
is particularly attractive because it obviates
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Amino acid sequence tags derived from GFP
and its relatives have been instrumental in the
exploration of intracellular distribution and
dynamics of proteins. No similar genetically
encoded RNA motif that exhibits intrinsic
fluorescence for labeling RNA has yet been
discovered, however. Three papers in this
issue of Nature Methods present powerful new
strategies for adopting fluorescent proteins as
reporters for the imaging of specific mRNAs
in live cells1–3. Valencia-Burton et al.1 and
Ozawa et al.2 describe two methods in which
mRNAs are revealed when they mediate
reconstitution of a fluorescent protein from
two nonfluorescent halves in bacteria and
human cells, respectively, and Haim et al.3
describe a scheme to tag yeast mRNAs on a
genomic scale with GFP reporters.
Figure 1 | Imaging endogenous mRNA in yeast. An RNA sequence corresponding to several MS2 coat
protein binding sites is introduced in the untranslated region of a gene in the genome of yeast. The
RNA product of the gene is rendered fluorescent when a fused GFP-MS2 coat protein coexpressed in the
same cell binds to the RNA.
Sanjay Tyagi is at the Public Health Research Institute, New Jersey Medical School, University of Medicine and
Dentistry of New Jersey, 225 Warren Street, Newark, New Jersey 07103, USA.
e-mail: [email protected]
the need to microinject probes into each cell
before imaging, and it allows the construction of stably expressing transgenic cell lines
and organisms. A successful example of this
approach involves tagging the mRNA with a
motif derived from RNA bacteriophage MS2
that tightly binds to the coat protein of MS2,
and then expressing this modified mRNA in
a cell that produces a fused GFP-MS2 coat
protein. The GFP-MS2 coat protein binds to
the RNA and renders it fluorescent5. Haim
et al. describe an approach in which any
endogenous yeast mRNA can be tagged and
imaged in this manner3 (Fig. 1). By increasing the number of MS2 coat protein–binding
sites on the RNA, it should even be possible
to detect single mRNA molecules in yeast, as
has been demonstrated previously in mammalian cells6.
A limitation that has vexed the GFP-MS2
approach is that the GFP reporter is always
fluorescent, whether or not it is bound to
the mRNA. Thus, it is possible to visualize
the mRNA only when the unbound reporter
protein is excluded from the cellular compartment where the mRNA is present, for
example, by restricting the GFP reporter
to the nucleus6, when the mRNA contains
many sites for the reporter protein to bind to,
or when the reporter protein is expressed in
exceedingly low amounts.
A solution to this problem was suggested
by the demonstration that GFP and related
proteins can be split into two halves in such
a way that neither half is fluorescent, and
the halves do not bind to each other when
coexpressed. If a pair of proteins that have an
affinity for each other are attached to these
GFP halves, the two complexes bind to each
other, allowing the GFP halves to associate
and fluoresce7. This strategy has been used
in a variety of contexts, including the screening of protein pairs that associate with each
other among the full complement of cellular proteins in vivo7. Notably, the protein
tags attached to the GFP halves do not even
have to bind directly to each other, they can
be brought in close proximity via a common
target, such as an mRNA molecule.
To adopt this ‘split GFP’ method for
mRNA detection, two RNA-binding proteins that can strongly and specifically bind
NATURE METHODS | VOL.4 NO.5 | MAY 2007 | 391
NEWS AND VIEWS
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C-terminal
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Kim Caesar
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Figure 2 | Split GFP complementation for tagging mRNA in live cells. (a) GFP fragments are fused to
a split RNA aptamer–binding protein, each half recognizing one side of an aptamer sequence inserted
downstream of the coding sequence. Fluorescence is reconstituted when these fusion proteins bind
the aptamer sequence. (b) Fluorescent protein fragments are fused to two RNA-binding proteins,
engineered to bind to adjacent endogenous RNA sequences. Binding of these fusions to the RNA
reconstitutes GFP fluorescence.
to different artificial RNA motifs are fused
with the two fragments of GFP, and the two
corresponding RNA motifs are introduced in
the target mRNA at adjacent sites. When the
engineered RNA is coexpressed in the cell,
the RNA-binding proteins fused to the GFP
fragments bind to their targets, and owing to
their proximity, promote reconstitution of
fluorescent GFP (Fig. 2). If the RNA is not
present, however, no fluorescence can be
seen in the cell. The efficacy of this approach
was first demonstrated by Rackham and
Brown, who introduced an MS2 coat protein–binding motif and a ‘zip code’ sequence
derived from β-actin mRNA into an artificial mRNA construct, and imaged it in cells
that also expressed GFP fragments fused to
392 | VOL.4 NO.5 | MAY 2007 | NATURE METHODS
the corresponding RNA-binding proteins8.
Valencia-Burton et al. used a single RNA
aptamer, instead of MS2 protein-binding
motifs, selected to bind to eukaryotic initiation factor 4A (eIF4A)1. eIF4A is a dumbbellshaped protein with two globular domains,
with each domain having a strong affinity to
one side of the same aptamer. The authors
split eIF4A into two parts, fused each part to
one GFP fragment and then expressed the
fusion proteins in Escherichia coli. When the
bacteria produced an mRNA containing the
aptamer, they exhibited GFP fluorescence
(Fig. 2a). With this approach researchers will
be able to study RNA dynamics and trafficking without interference from background
signals.
Ozawa et al. took a different, but equally
elegant, approach in human cells2. They used
human protein PUMILIO1 (a homolog of
the Drosophila melanogaster protein called
Pumilio), which has the distinction of being
the only known RNA-binding protein that
binds to stretches of RNA in a sequence-specific manner9. Furthermore, the sequence of
this protein can be modified to alter its target
sequence specificity in a predictable manner10.
Thus, Ozawa et al.2 were able to use two varieties of PUMILIO1 to bind to two adjacent eightnucleotide stretches on an endogenous mRNA
(Fig. 2b). Their results show that split fragments
of GFP or a yellow fluorescent protein called
Venus fused to the PUMILIO1 variants complemented in the presence of the target RNA, and
the fluorescence was restored in mitochondria
of HeLa cells, where Pumilio’s target mRNA
is normally expressed. A major attraction of
this approach is that a pair of proteins can be
designed for the detection of any natural mRNA
without altering the RNA sequence.
These methods not only solve the problem
of background fluorescence stemming from
unbound GFP reporters, they point to ways
in which more than one RNA species can
be visualized and tracked in the same cell.
Fluorescent proteins of different hues can
be split and complemented with each other,
resulting in combinatorial possibilities for the
generation of new colors for multiplexing7.
The results reported in these papers demonstrate the imaging of mRNAs in bacteria, yeast
and higher eukaryotes, thus opening up new
avenues for exploring the rich biology of RNA
across the entire evolutionary continuum.
ACKNOWLEDGMENTS
The author’s research is supported by US National
Institutes of Health grant GM-070357.
COMPETING INTERESTS STATEMENT
The author declares no competing financial interests.
1.
Valencia-Burton, M., McCullough R.M., Cantor,
C.R. & Broude, N.E. Nat. Methods 4, 421–427
(2007).
2. Ozawa, T., Natori, Y., Sato, M. & Umezawa, Y. Nat.
Methods 4, 413–419 (2007).
3. Haim, L., Zipor, G., Aronov, S. & Gerst J.E. Nat.
Methods 4, 409–412 (2007).
4. Bratu, D.P., Cha, B.-J., Mhlanga, M.M., Kramer,
F.R. & Tyagi, S. Proc. Natl. Acad. Sci. USA 100,
13308–13313 (2003).
5. Bertrand, E. et al. Mol. Cell 2, 437–445 (1998).
6. Fusco, D. et al. Curr. Biol. 13, 161–167 (2003).
7. Kerppola, T.K. Nat. Rev. Mol. Cell Biol. 7, 449–456
(2006).
8. Rackham, O. & Brown, C.M. EMBO J. 23, 3346–
3355 (2004).
9. Wang, X., McLachlan, J., Zamore, P.D. & Hall, T.M.
Cell 110, 501–512 (2002).
10. Cheom-Gil, C., Traci, M. & Tanaka, H. Proc. Natl.
Acad. Sci. USA 103, 133635–133639 (2006).