DOI: 10.1126/science.1195691
, 1081 (2011);331Science
et al.Keren Nevo-Dinur
E. coliTranslation-Independent Localization of mRNA in
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Translation-Independent
Localization of mRNA in E. coli
Keren Nevo-Dinur, Anat Nussbaum-Shochat, Sigal Ben-Yehuda, Orna Amster-Choder*
Understanding the organization of a bacterial cell requires the elucidation of the mechanisms
by which proteins localize to particular subcellular sites. Thus far, such mechanisms have
been suggested to rely on embedded features of the localized proteins. Here, we report that
certain messenger RNAs (mRNAs) in Escherichia coli are targeted to the future destination of their
encoded proteins, cytoplasm, poles, or inner membrane in a translation-independent manner.
Cis-acting sequences within the transmembrane-coding sequence of the membrane proteins are
necessary and sufficient for mRNA targeting to the membrane. In contrast to the view that
transcription and translation are coupled in bacteria, our results show that, subsequent to their
synthesis, certain mRNAs are capable of migrating to particular domains in the cell where
their future protein products are required.
T
argeting of specific mRNAs to different
subcellular domains is generally believed
to occur only in eukaryotes, where the synthesis
and processing of mRNA is compartmentalized
between nucleus and cytoplasm (1–3).
In bacteria, gene transcription and translation
are not thought to be compartmentalized, and
conventional wisdom holds that translation is
coupled to mRNA synthesis (4). Consequently,
protein localization is believed to be governed
by intrinsic features of proteins and not of their
mRNA transcripts (5–7). Accordingly, several
bacterial mRNAs have been shown to localize
near the site of their synthesis on the genome
(8). On the other hand, the transcription machinery
and the ribosomes occupy different subcellular
regions within different bacterial cells
(9, 10), raising the possibility that mature mRNAs
can move from the nucleoid to other cellular sites
where they are translated. Here, we report the
existence of bacterial mechanisms for subcellular
protein localization that operate at the level
of mRNA targeting in a translation-independent
manner.
We followed the location of cat and lacY transcripts,
encoding the cytoplasmic chloramphenicol
acetyltransferase and the membrane-bound
lactose permease, respectively (11, 12), in living
Escherichia coli cells by cloning six copies of the
binding sequence of phage MS2 coat protein
(6xbs) upstream of these genes. We visualized
the resulting 6xbs-tagged cat and lacY transcripts
(cat6xbs and lacY6xbs, respectively) by fluorescence
microscopy in cells expressing the MS2 coat
protein fused to green fluorescent protein (MS2GFP)
(fig. S1) (13). We observed the cat transcripts
in the cytoplasm in a helixlike pattern (Fig. 1A,
a, and fig. S2), reminiscent of the helical RNA
structures documented previously in E. coli (14).
In contrast, we detected lacY transcripts preferentially
at or near the cytoplasmic membrane
(Fig. 1A, b). In cells lacking 6xbs-tagged transcripts,
MS2-GFP was evenly distributed throughout
the cytoplasm (Fig. 1A, c), whereas the
distribution of short transcripts, composed of the
6xbs, was similar to cat (fig. S3). We also examined
the subcellular localization of cat and
lacY transcripts by cellular fractionation followed
by reverse transcription and polymerase
chain reaction amplification (RT-PCR), resulting
in the detection of cat and lacY transcripts in the
cytosolic and membrane fractions, respectively
(Fig. 1B, a and b). We used real-time PCR to
quantify the enrichment in cat and lacY transcripts
in the cytosolic and membrane fractions, respectively
(fig. S4). Taken together, each of these
transcripts localizes to the compartment where
its protein product functions.
To examine the subcellular localization of transcripts
of an operon that encodes both membrane
and soluble proteins, we introduced the
6xbs in various locations within the bgl operon
of E. coli that encodes the BglG transcription
factor, the BglF membrane-bound sugar permease,
and the soluble phospho-b-glucosidase,
BglB. We then confirmed functionality of all 6xbstagged
transcripts [see supporting online material
(SOM) (15)]. When the 6xbs were introduced
immediately upstream of the chromosomal bglF
gene, the MS2-GFP protein was observed at the
cell membrane (Fig. 1A, d). In contrast, when
the chromosomal bglF gene was replaced by
cat6xbs, the MS2-GFP displayed a helical, awayfrom-the-membrane
distribution, the same as
that of plasmid-encoded cat (Fig. 1A, e). We noticed
localization patterns similar to the ones
obtained in live cells when we used fluorescence
in situ hybridization (FISH) to detect bglF and
cat transcripts (Fig. 1D and fig. S5). To examine
the correlation between the localization of the
bglF transcript and the BglF protein, mCherry
was fused to bglF6xbs. We observed the BglFmCherry
fusion protein around the cell membrane
(Fig. 1C, b), which is similar to the MS2-GFP
localization pattern (Fig. 1C, a and c) and different
from the localization of mCherry mRNA and
protein when not fused to BglF (fig. S6). In fact,
all truncated transcripts of the bgl operon that
included bglF localized to the membrane (Fig.
1A, g, h, j, and k), including transcripts of bglF
expressed as a monocistron (Fig. 1A, f). This
pattern of localization did not depend on whether
the 6xbs were introduced adjacent to bglF
(Fig. 1A, f, g, and j), bglB (Fig. 1A, h), or bglG
(Fig. 1A, k). However, transcripts expressing
only the soluble BglB protein were distributed
similarly to cat transcripts (Fig. 1A, i), whereas
those containing only the bglG gene were detected
near the poles (Fig. 1A, l), similar to the
localization pattern of the BglG protein in cells
not expressing BglF (16). Cellular fractionation
followed by RT-PCR confirmed that all transcripts
containing the membrane protein–encoding
bglF gene localized to the membrane, whereas
transcripts encoding only the soluble BglB protein
were present in the cytosol, and those encoding
the polar BglG were detected in the membrane
and in the cytosolic fractions (Fig. 1B, c to k).
Thus, localization of the monocistronic transcripts
of each bgl gene correlates with subsequent localization
of the respective protein products. However,
the membrane-encoding cistron is dominant
to the hydrophilic-encoding cistron in determining
localization of multicistronic mRNAs. Conversely,
a bicistronic mRNA, which consists of
bglG and cat6xbs, exhibited the localization pattern
of both partners—that is, helical with accumulation
near the poles (Fig. 1A, m, and fig.
S7, a to c).
To determine if targeting is a property of the
mRNA, we uncoupled transcription and translation
by treating the cells with vast excess of
kasugamycin or chloramphenicol, which inhibit
initiation or elongation of translation, respectively
(17, 18). These treatments resulted in complete
inhibition of bglF mRNA translation (fig. S8),
but did not affect targeting of bglF transcripts to
the membrane (Fig. 2, B and F). Treatment with
each antibiotic had no effect on the distribution
of MS2-GFP itself (Fig. 2, A and E). Chloramphenicol
was reported to slightly increase stability
of certain mRNAs (19), but we saw no substantial
difference in the half-life of bglF transcripts in
treated and untreated cells (fig. S9). Of note, the
half-life of bglF transcripts is shorter than that of
most E. coli mRNAs (20) and considerably
shorter than the duration of treatment with both
antibiotics. Hence, most, if not all, bglF transcripts
observed after antibiotic treatment were
new untranslated transcripts. Treatment with the
transcription-inhibiting drug rifampicin, with or
without kasugamycin or chloramphenicol, resulted
in a lack of bglF6xbs transcripts and, hence,
in even spreading of MS2-GFP molecules in the
cell (fig. S10). The distribution of bglG6xbs or
cat6xbs transcripts was also not affected by the
translation-inhibiting antibiotics (Fig. 2, C, D,
and G, and fig. S11). Therefore, these mRNAs
seem to harbor the information that determines
their nonrandom localization in the cell by a
Department of Microbiology and Molecular Genetics, Institute
for Medical Research Israel-Canada, The Hebrew University
Faculty of Medicine, Post Office Box 12272, Jerusalem
91120, Israel.
*To whom correspondence should be addressed. E-mail:
amster@cc.huji.ac.il
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translation-independent mechanism. To reinforce
this conclusion, we aimed at uncoupling transcription
and translation by a different approach.
Mutating the first codon of bglF yielded transcripts
that were targeted to the membrane (Fig.
3A), but some translation from a downstream
start codon was detected (fig. S12). To completely
inhibit protein synthesis, we introduced a “5translational
blockage” (21), which consisted of
12 consecutive rare leucine codons, near the 5′
end of the bglF gene. The resultant transcript
was readily detected by RT-PCR, but as
expected, its translation was blocked (fig. S13).
Noticeably, this untranslated bglF mRNA was
viewed near the membrane (Fig. 3B), demonstrating
once again that membrane recruitment
of the mRNA is translation-independent.
To characterize the mRNA element that targets
bglF transcripts to the membrane, we fused
the 6xbs to truncated bglF derivatives that encode
either the hydrophilic IIBbgl
domain or the
Fig. 1. Subcellular localization
of mRNA transcripts
correlates with
subsequent localization
of their protein products.
(A) Fluorescence microscopy
images of cells expressing
MS2-GFP (green)
and transcripts containing
or lacking binding
sites for MS2-GFP. “6xbs”
after a gene’s name denotes
that the MS2 binding
sites were introduced
immediately next to this
gene. Cells shown in (l)
were supplemented with
the fluorescent membrane
stain FM4-64. Transcripts
were plasmid-encoded,
unless otherwise stated.
Scale bar, 1 mm. (B) Cells
deleted for the bgl operon
and transformed with plasmids
encoding the genes
listed on the right were
fractionated as described
in the SOM (see fig. S17
for verification of successful
fractionation) (15).
Transcripts, expressed as
mono- or multicistronic,
present in the different
fractions were monitored
by RT-PCR. Purple indicates
those genes whose
sequence was amplified.
In (h) and (k), the junction
region between the
genes in purple was amplified.
mem, membrane;
cyto, cytosol. (C) (a and
b) Fluorescence microscopy
images of cells expressing
MS2-GFP (green) and
a BglF-mCherry fusion protein
(red), whose transcript
contains the 6xbs. (c) An
overlay of the signals.
Scale bar, 1 mm. The possibility
of false detection
due to fluorescence leakage
between the filters
was ruled out. The white
arrows point at a cell in
which the 6xbs-tagged
bglF-mCherry transcripts
are detected (a) and the BglF-mCherry protein is not (b) (see also the results in fig. S18). (D) Chromosome-encoded bglF6xbs (a) or cat6xbs (b) transcripts were
detected by FISH, as described in the SOM (15), and DNA was stained with 4´,6´-diamidino-2-phenylindole (see fig. S5) (15). Scale bar, 1 mm.
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hydrophobic IICbgl
domain [designated the B
and C domains, respectively, hereafter (22)] and
followed their subcellular localization. Transcripts
encoding the soluble B domain were observed
in the cytoplasm (Fig. 4A, a), in accord
with localization of the encoded protein (Fig.
4B), whereas those encoding the membrane C
domain were detected at the membrane (Fig.
4A, b). Therefore, the membrane-targeting information
is located within the sequence that encodes
the membrane-spanning domain. Transcripts
of a bglF6xbs variant that contains a stop codon
before the end of the first domain, thereby yielding
a full-length bglF transcript that encodes
only the soluble B domain (termed B22; fig.
S14), were observed at the membrane (Fig. 4A,
c), substantiating the notion that the cis-acting
sequence for membrane targeting is contained
within the transcript. RT-PCR after cell fractionation
confirmed the microscopy observations (Fig.
4C). Taken together, targeting of bglF transcripts
to the membrane requires the region that encodes
the membrane domain, but translation of this
region is not necessary. Further dissection of the
sequences required for transcripts localization
demonstrated that the sequence encoding the
first two transmembrane helices of BglF is sufficient
for membrane targeting of the transcripts
(Fig. 4A, d), whereas the region encoding the
N-terminal RNA-binding domain of BglG is sufficient
for mRNA polar targeting (fig. S7, d to f).
The results presented here show that mechanisms
for targeting mRNAs to subcellular locations,
where their protein products are required,
exist in bacteria. Previously, mRNA features were
proposed to be involved in targeting proteins to
the type III secretion system in Gram-negative
bacteria (23). mRNA targeting might be tied to
processes other than localized translation, for
instance, mRNA degradation. The latter possibility
is in accord with previous reports showing
that components of the E. coli mRNA degradosome
associate with the membrane and assemble
into helical filaments (24, 25). Our study
also establishes the existence of a translationindependent
RNA transport mechanism in bacteria.
By and large, mRNA localization can be
achieved by facilitated diffusion in the cytoplasm
or by active transport along cytoskeletal
filaments (26, 27). The short half-life of the bglF
transcripts suggests that an active mechanism is
involved in targeting them to the membrane.
The finding of inner membrane-bound ribosomes
in E. coli, which are actively engaged in translation
(28), indicates that the set-up for translation
of transcripts that encode membrane proteins exists.
Regarding the zip codes that target mRNAs
to the membrane, a recent analysis suggested that
uracils, which are highly represented in transcripts
that encode integral membrane proteins,
might serve as a physiologically relevant signature
to this group of mRNAs (29). This evolutionarily
conserved U richness might serve as a
zip code that attracts proteins, including ribosomal
proteins, which bring the transcripts to the memFig.
2. Transcripts localization is
not affected by inhibition of translation.
(A to G) Fluorescence microscopy
images of cells expressing the
MS2-GFP protein (green) and bglGbglF
transcripts lacking binding sites
for MS2-GFP [(A) and (E)], bglGbglF6xbs
[(B) and (F)], bglG6xbs [(C)
and (G)], or cat6xbs (D) transcripts.
The cells were treated for 10 min
with 10mg/ml kasugamycin [(A) to
(D)] or 250mg/ml chloramphenicol
[(E) to (G)] before microscopy. Scale
bar, 1 mm.
Fig. 3. Membrane localization
of bglF transcripts
is not affected by translational
blockage. Fluorescence
microscopy images
of cells expressing the MS2GFP
protein (green) and
6xbs-tagged transcripts of
bglF alleles, which have
either a mutated start codon
(A) or 12 consecutive
rare leucine codons
(CUA) near the 5′ end
(B). Scale bar, 1 mm.
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brane. Indeed, an mRNA encoding the rhomboid
1 transmembrane protein from Drosophila melanogaster
still localized predominantly to the membrane
in E. coli cells (fig. S15). Finally, our
results imply that mRNA targeting is in tight
correlation with the requirements for complex
formation. Specifically, bglG mRNA is targeted
to the membrane or to the poles, depending on
whether it is expressed with or without bglF,
respectively. This is in full agreement with the
localization of the BglG protein, which associates
with BglF at the membrane (30) or with the general
PTS proteins at the poles (16), depending on
environmental conditions.
References and Notes
1. E. Lécuyer et al., Cell 131, 174 (2007).
2. D. St. Johnston, Nat. Rev. Mol. Cell Biol. 6, 363 (2005).
3. C. E. Holt, S. L. Bullock, Science 326, 1212 (2009).
4. I. Iost, M. Dreyfus, EMBO J. 14, 3252 (1995).
5. A. Janakiraman, M. B. Goldberg, Trends Microbiol. 12,
518 (2004).
6. G. Ebersbach, C. Jacobs-Wagner, Trends Microbiol. 15,
101 (2007).
7. L. Shapiro, H. H. McAdams, R. Losick, Science 326,
1225 (2009).
8. P. Montero Llopis et al., Nature 466, 77 (2010).
9. P. J. Lewis, S. D. Thaker, J. Errington, EMBO J. 19,
710 (2000).
10. T. A. Azam, S. Hiraga, A. Ishihama, Genes Cells 5,
613 (2000).
11. M. W. Chen, V. Nagarajan, J. Bacteriol. 175, 5697
(1993).
12. L. Guan, H. R. Kaback, Annu. Rev. Biophys.
Biomol. Struct. 35, 67 (2006).
13. I. Golding, E. C. Cox, Proc. Natl. Acad. Sci. U.S.A. 101,
11310 (2004).
14. M. Valencia-Burton et al., Proc. Natl. Acad. Sci. U.S.A.
106, 16399 (2009).
15. Materials and methods, as well as other supporting
material, are available on Science Online.
16. L. Lopian, Y. Elisha, A. Nussbaum-Shochat,
O. Amster-Choder, EMBO J. 29, 3630 (2010).
17. F. Schluenzen et al., Nat. Struct. Mol. Biol. 13,
871 (2006).
18. A. S. Weisberger, Annu. Rev. Med. 18, 483 (1967).
19. U. Lundberg, G. Nilsson, A. von Gabain, Gene
72, 141 (1988).
20. J. A. Bernstein, A. B. Khodursky, P. H. Lin, S. Lin-Chao,
S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 99, 9697
(2002).
21. E. Goldman, A. H. Rosenberg, G. Zubay, F. W. Studier,
J. Mol. Biol. 245, 467 (1995).
22. O. Amster-Choder, Curr. Opin. Microbiol. 8, 127 (2005).
23. J. A. Sorg, N. C. Miller, O. Schneewind, Cell. Microbiol. 7,
1217 (2005).
24. A. Taghbalout, L. Rothfield, Proc. Natl. Acad. Sci. U.S.A.
104, 1667 (2007).
25. V. Khemici, L. Poljak, B. F. Luisi, A. J. Carpousis,
Mol. Microbiol. 70, 799 (2008).
26. K. Czaplinski, R. H. Singer, Trends Biochem. Sci.
31, 687 (2006).
27. I. M. Palacios, Semin. Cell Dev. Biol. 18, 163 (2007).
28. A. A. Herskovits, E. Bibi, Proc. Natl. Acad. Sci. U.S.A. 97,
4621 (2000).
29. J. Prilusky, E. Bibi, Proc. Natl. Acad. Sci. U.S.A. 106,
6662 (2009).
30. L. Lopian, A. Nussbaum-Shochat, K. O’Day-Kerstein,
A. Wright, O. Amster-Choder, Proc. Natl. Acad. Sci. U.S.A.
100, 7099 (2003).
31. We acknowledge members of S.B.-Y.’s group for help with
fluorescence microscopy, members of O.A.-C.’s group
for stimulating discussions, and E. Bibi and E.S.
Bochkareva for technical advice on cell fractionation.
We thank the following individuals for their gifts:
I. Golding for pIG-K33, R. H. Singer for pSL-MS2 6, and
Z. Livneh for purified single-stranded DNA binding protein
(SSB) and anti-SSB serum. We are grateful to
A. Wright, E. Bibi, and R. Losick for critically reading
our manuscript. This research was supported by a
research grant (awarded to O.A.-C.) by the Israel
Science Foundation founded by the Israel Academy of
Sciences and Humanities. S.B.-Y. is supported by the
European Research Council starting grant (209130).
Supporting Online Material
www.sciencemag.org/cgi/content/full/331/6020/1081/DC1
Materials and Methods
Figs. S1 to S18
Table S1
References
27 July 2010; accepted 13 January 2011
10.1126/science.1195691
Fig. 4. Membrane localization
of bglF transcripts
requires the membrane
domain-encodingsequence.
(A) Fluorescence microscopy
images of cells expressing
the MS2-GFP
protein (green) and 6xbstagged
truncated bglF
transcripts encoding: (a)
the soluble B domain;
(b) the membrane-bound
C domain; (c) the mutant
B22 allele, which yields
a full-length transcript
but a truncated B domain
due to a stop codon; and
(d) the B domain and the
first two transmembrane
helices of the C domain.
The bglF transcripts and
their respective protein
products are schematically
shown. stop, stop codon;
b, bases; aa, amino
acids. Scale bar, 1 mm.
(B) Fluorescence microscopy
images of cells
expressing MS2-GFP
(green) and the B domain
of BglF fused to mCherry (red), whose transcript contains the 6xbs. Scale bar, 1 mm. (C) Cell fractionation and detection of transcripts by RT-PCR as described in
Fig. 1B. (a), (b), and (c) are as in (A). The bglF sequence amplified in each case is in purple.
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