BRIEF COMMUNICATIONS
A genomic integration
method to visualize
localization of
endogenous mRNAs
in living yeast
Liora Haim, Gadi Zipor, Stella Aronov &
Jeffrey E Gerst
mRNA localization may be an important determinant for protein
localization. We describe a simple PCR-based genomic-tagging
strategy (m-TAG) that uses homologous recombination to insert
binding sites for the RNA-binding MS2 coat protein (MS2-CP)
between the coding region and 3¢ untranslated region (UTR) of
any yeast gene. Upon coexpression of MS2-CP fused with GFP,
we demonstrate the localization of endogenous mRNAs (ASH1,
SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).
Local mRNA translation is involved in cell-fate determination,
polarization and morphogenesis in eukaryotes1–3. Thus, specialized
tools to monitor mRNA trafficking in individual cells are needed.
The localization of endogenous mRNA has been examined using
fluorescence in situ hybridization (FISH) with labeled RNA probes
but requires formaldehyde-fixed cells or tissues. Plasmid-based
expression systems are used to exogenously express mRNAs bearing
binding sites for RNA-binding proteins (such as MS2-CP) and,
when coexpressed with an RNA-binding protein fused with GFP
(such as MS2-CP–GFP), allow for mRNA localization in living
cells4,5. Plasmid expression, however, results in higher-than-
endogenous mRNA levels, which could result in mRNA mislocalization. Endogenous mRNAs in living cells have been tracked
using fluorescent hybridization probes, but these techniques are
difficult to use and the signals are transient6. Thus, a simple
technique for the sustained visualization of endogenous mRNA
in vivo is required.
PCR-based strategies for genomic tagging in yeast, via homologous recombination, have yielded deletion libraries7 as well as
GFP- and epitope-tagged protein expression libraries8,9. To develop
a similar resource for the systematic mapping of mRNA localization, we created an integration construct that can be amplified by
PCR using oligonucleotides bearing homology to the desired site of
chromosomal insertion. The insertion cassette (Fig. 1) contains 12
MS2-CP binding sites (MS2 loops; MS2L) cloned downstream to
the Schizosaccharomyces pombe his5+ selectable marker, which
confers growth in medium lacking histidine, flanked by loxP sites.
The latter are used for Cre recombinase–mediated excision of the
marker after integration and cre expression10. This places MS2-CP
binding sites downstream of the stop codon and upstream of
the 3¢ UTR, the latter often necessary for mRNA targeting in
eukaryotes1–3. For example, we recently showed that 3¢ UTRs
facilitate the trafficking of mRNAs encoding polarity or secretion
a
ORF
MS2L (X2)
Marker
loxP
Template 5′
loxP
3′
3′ UTR
Oligo 2 (Reverse)
PCR
PCR
product
b
Figure 1 | A schematic representation of the MS2 loop genomic-tagging
strategy (m-TAG). (a) Forward and reverse oligonucleotide primers having
identity to the coding region (including stop codon) and 3¢ UTR of a given
gene (open reading frame; ORF), respectively, are used to amplify a template
cassette by PCR. The template cassette contains 12 MS2 loop sequences
(MS2L) and a selectable marker (Sphis5+) flanked by loxP sites. (b) PCR
amplification yields the indicated product, which is transformed into yeast.
(c) Homologous recombination results in integration into the gene between
the coding region and 3¢ UTR. (d) cre recombinase expression results in
excision of the selectable marker located between the loxP sites, leaving one
loxP site and MS2L juxtaposed between the coding region and the 3¢ UTR.
(e) After verification of integration and marker excision by PCR analysis and
sequencing, cells are transformed with a plasmid encoding MS2-CP–GFP(3)
to visualize mRNA localization.
Oligo 1 (Forward)
ORF
loxP
Marker
ORF
Genome
Homologous
c
ORF
loxP
Marker
Cre
d
ORF
loxP
MS2-CP–GFP
e
mRNA
ORF
loxP
loxP
3′ UTR
3′ UTR
recombination
loxP
3′ UTR
recombination
3′ UTR
expression
3′ UTR
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. Correspondence should be addressed to J.E.G. ([email protected]).
RECEIVED 21 DECEMBER 2006; ACCEPTED 16 MARCH 2007; PUBLISHED ONLINE 8 APRIL 2007; DOI:10.1038/NMETH1040
NATURE METHODS | VOL.4 NO.5 | MAY 2007 | 409
BRIEF COMMUNICATIONS
ASH1
CLN2
Budding cells (%)
Wild type
20
60
50
15
40
10
30
0
she2 ∆
25
Merge
80
ASH1INT
70
Relative value
20
60
50
15
40
10
30
20
5
Budding cells (%)
MS2-CP
–GFP (×3)
Time (min)
MS2-CP–GFP (×3)
GFP-NLS-Cln2
10
0
b
DIC
ash1∆
Wild type
20
5
d
80
70
Budding cells (%)
Wild type
MS2-CP
–GFP (×3)
25
Wild type
c
Merge
ASH1INT
GFP
Relative value
DIC
MS2-CP
–GFP (×2)
a
10
0
S
Early
G2-M
Late
G2-M
0
10 20 30 40 50 60 75 90 105 120
0
Time (min)
Figure 2 | Visualization of endogenous ASH1 mRNA localization in vivo. (a) Fluorescence and light microscopy images of ASH1::loxP::MS2L::ASH13¢UTR cells
in early G2-M phase, transformed with plasmids encoding the indicated constructs in representative wild-type or ASH1::loxP::MS2L::ASH13¢UTR she2D cells.
DIC, differential interference contrast image; GFP, GFP fluorescence image. (b) ASH1::loxP::MS2L::ASH13¢UTR cells expressing MS2-CP–GFP(3) in different
stages of the cell cycle (merged DIC and GFP fluorescence microscopy). (c) Relative values of ASH1 and CLN2 mRNA levels in synchronized wild-type and
ASH1::loxP::MS2L::ASH13¢UTR (ASH1INT) cells measured by real-time PCR (Supplementary Methods), as a function of time. The percentage of budded cells
was determined by microscopy. Error bars indicate the s.d. obtained from duplicate samples in a representative experiment. (d) Wild-type, ash1D, and
ASH1::loxP::MS2L::ASH13¢UTR (ASH1INT) cells were transformed with a plasmid expressing GFP tagged with a nuclear localization signal (NLS) and PEST
degradation signal from Cln2 (GFP-NLS-Cln2). Representative cells showing the localization of GFP-NLS-Cln2 are presented in DIC, GFP fluorescence
microscopy and the merged DIC-fluorescence images. Scale bars, 1 mm.
factors to the incipient bud11. As other genomic tagging strategies
use integration constructs that invariably dissociate the 3¢ UTR
from the coding sequence, that is, upon insertion of GFP and the
kanamycin-resistance cassette at the 3¢ end of genes9, the resulting
mRNAs (and translated proteins) may be mislocalized. Thus,
excision of the selectable marker is necessary to position the
3¢ UTR close to the coding region (Fig. 1).
As integration and Cre-mediated excision can be monitored by
PCR, we determined whether this gene-tagging technique is feasible
using ASH1 mRNA, which has been shown to localize to the bud
tip using FISH and plasmid-based in vivo labeling4,5,12,13. During
budding, ASH1 mRNA is exported to daughter cells, wherein local
translation prevents mating-type switching3,12,13. We used PCR to
amplify the loxP::Sphis5+::loxP::MS2L cassette (Fig. 1) using a
forward oligonucleotide complementary to the 3¢ end of the
ASH1 coding region (including stop codon) and a sequence
upstream of the first loxP site, and a reverse oligonucleotide
complementary to the 5¢ end of the ASH1 3¢ UTR and a nonrepetitive sequence at the 3¢ end of MS2L (Supplementary Table 1
online). Transformation into wild-type yeast (BY4741 cells; see
Supplementary Table 2 online for yeast strains) led to colony
formation on medium lacking histidine. DNA extraction and PCR
amplification using oligonucleotides complementary to the ASH1
coding region and the loxP::Sphis5+::loxP::MS2L::ASH13¢UTR
sequence revealed proper integration by gel electrophoresis
and DNA sequencing (Supplementary Fig. 1 online). ASH1::loxP::
Sphis5+::loxP::MS2L::ASH13¢UTR integration had a frequency of
B60%. Next, we used cre expression from a galactose-inducible
promoter to excise his5+ to yield ASH1::loxP::MS2L::ASH13¢UTR
410 | VOL.4 NO.5 | MAY 2007 | NATURE METHODS
(ASH1INT) cells (Supplementary Fig. 1). Marker excision and
histidine auxotrophy occurred at a frequency of B100%. After
Cre-mediated recombination we extracted total RNA and, after
reverse-transcription PCR, sequenced it to verify the presence of
MS2L and the 3¢ UTR in the transcript (Supplementary Fig. 1).
Thus, a PCR-based strategy can efficiently integrate viral RNA
binding sites into the yeast genome.
To visualize ASH1 mRNA localization in the ASH1INT strain, we
expressed MS2-CP–GFP from the inducible MET25 promoter. After
induction (1 h) in medium lacking methionine, we examined the
cells for fluorescence-labeled granular mRNA (gRNA)4,5,14. The
pattern of labeled mRNA (previously observed for different genes,
organisms and using different labeling techniques) is typically
granular, and large granules containing multiple mRNAs have
been observed, probably as a result of the cumulative association
of smaller granules accumulating at a given site. We could not, however, detect significant amounts of gRNA, perhaps because endogenous mRNA levels are substantially lower than those obtained
using plasmid-based expression11. Thus, we created double- and
triple-GFP–tagged MS2-CP fusions and expressed them in ASH1INT
yeast (Fig. 2a). Expression of MS2-CP–GFP(2) led to the appearance of granules in 3% of cells (n ¼ 200), whereas the expression of
MS2-CP–GFP(3) led to granules in 19% of cells (n ¼ 200).
Endogenous ASH1 gRNA concentrated at the bud tip in 98% of
small- and medium-budded cells (S–early G2–M; n ¼ 91) and at the
bud neck in 82% of large-budded cells (late G2–M; n ¼ 110) in cells
expressing MS2-CP–GFP(3) (Fig. 2b), as seen with exogenous
ASH1 mRNA4,5,14. ASH1 gRNAs reached B200 nm in size, as seen
before4,5,11,14, and contained 2.4–15.6 individual ASH1INT mRNAs
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a
DIC
MS2-CP–GFP(×3)
c
Merge
Wild type
SRO7
pex3∆
PEX3INT
b
DIC
MS2-CP–GFP(×3)
Sec63-RFP
Merge
d
DIC
MS2-CP–GFP(×3)
PEX3
Oxa1-RFP
Merge
OXA1
e
Wild type
oxa1∆
OXA1INT
Figure 3 | Visualization of other endogenous yeast mRNAs in vivo. (a) Representative DIC and fluorescence microscopy images of integrated
SRO7::loxP::MS2L::SRO73¢UTR cells, transformed with a plasmid expressing MS2-CP–GFP(3). (b) Representative fluorescence and DIC microscopy images of
integrated PEX3::loxP::MS2L::PEX33¢UTR cells, transformed with plasmids expressing MS2-CP–GFP(3) and Sec63-RFP (an endoplasmic reticulum marker). Note the
juxtaposition or overlap between PEX3 gRNA and either nuclear or cortical endoplasmic reticulum. (c) Wild-type, pex3D and PEX3::loxP::MS2L::PEX33¢UTR (PEX3INT)
cells were grown to mid-log phase on glucose-containing medium, serially diluted, plated onto solid synthetic medium containing oleate as a carbon source and
grown for 48 h at 26 1C. Note that functional Pex3 is necessary for growth on oleate. (d) Representative DIC and fluorescence microscopy images of integrated
OXA1::loxP::MS2L::OXA13¢UTR cells, transformed with plasmids expressing MS2-CP–GFP(3) and Oxa1-RFP (a mitochondrial marker). Scale bars, 1 mm. (e) Wildtype, oxa1D and OXA1::loxP::MS2L::OXA13¢UTR (OXA1INT) cells were grown to mid-log phase on glucose-containing medium, serially diluted, plated onto solid
synthetic medium containing glycerol as a carbon source and grown for 48 h at 26 1C. Note that functional Oxa1 is necessary for growth on glycerol.
(n ¼ 30), as calculated using an integrated 128-mer lac operator
GFP fluorescence–quantification system (see Supplementary
Methods online). We observed no granules in MS2-CP–GFP(3)expressing cells lacking integrated MS2L (data not shown).
To verify that MS2L integration did not alter mRNA transport, we
examined ASH1 mRNA localization in cells lacking SHE2, an RNAbinding protein necessary for localization to the bud tip3. ASH1
mRNA mislocalized to the mothers in 460% of ASH1INT she2D
cells examined (n ¼ 100; Fig. 2a). Thus, the requirements for ASH1
localization are unchanged upon MS2L integration. Next we examined whether cell cycle–dependent ASH1 expression was altered,
using real-time PCR. ASH1 mRNA levels rose precipitously within
20 min of release from a-factor arrest; peaked within 50 min; and
were concomitant with budding in both wild-type and ASH1INT
cells (Fig. 2c). ASH1 mRNA levels then declined substantially, in
contrast to CLN2 mRNA (Fig. 2c), which increases as cells re-enter
G1 (ref. 15). Thus, ASH1 mRNA expression was unaltered upon
MS2L integration. Finally, we examined the effect of MS2L integration on Ash1 protein function. We expressed GFP tagged with Cln2
degradation and nuclear localization sequences under the control of
the Ash1-regulated HO promoter. This reporter localizes only to
mothers during budding in wild-type cells, but to both mothers and
daughters in cells lacking ASH1 (ref. 16). The reporter localized only
to mothers in 89% and 87% of wild-type and ASH1INT cells,
respectively, and to both mothers and daughters in 73% of control
ash1D yeast (n ¼ 100; Fig. 2d). Thus, MS2L integration does not
alter the ability of Ash1 to restrict expression from the HO promoter.
To determine whether other mRNAs can be localized using
m-TAG, we examined the localization of SRO7 mRNA, which
encodes an exocytosis factor17. We previously demonstrated that
SRO7 mRNA undergoes polarized transport, along with cortical
ER, to the bud tip11. Endogenous SRO7 gRNA localized to bud
tips in 450% of small-budded SRO7::loxP::MS2L::SRO73¢UTR
(SRO7INT) cells expressing MS2-CP–GFP(3) (n ¼ 100; Fig. 3a).
We observed single granules in 72% of cells and two granules in
25% of cells (n ¼ 100). To determine whether MS2L integration
affects Sro7 function, we examined whether deletion of the SRO7
homolog, SRO77, in SRO7INT yeast results in cold-sensitive growth,
as cells lacking both genes grow poorly at low temperatures17.
The deletion of SRO77 in SRO7INT cells did not result in coldsensitivity, unlike in control sro7D cells (Supplementary Fig. 2
online). Thus, Sro7 remains functional upon MS2L integration into
the SRO7 locus.
Next we examined PEX3 mRNA, which encodes a peroxin that
localizes to the endoplasmic reticulum upon translation and
facilitates peroxisome assembly18. We created a PEX3::loxP::MS2L::
PEX33¢UTR strain (PEX3INT) and examined the localization of PEX3
mRNA upon MS2-CP–GFP(3) expression. In contrast to ASH1
NATURE METHODS | VOL.4 NO.5 | MAY 2007 | 411
BRIEF COMMUNICATIONS
or SRO7 gRNA, PEX3 gRNA was nonpolarized (16% bud-labeling;
n ¼ 50) and colocalized/juxtaposed with Sec63-RFP, an endoplasmic reticulum marker, in 80% of cells (n ¼ 50; Fig. 3b). In
contrast, few cells had PEX3 gRNA juxtaposed with a mitochondrial
marker, Oxa1-RFP (14%; n ¼ 50). We observed multiple (2–6)
PEX3 gRNAs in B50% of cells (n ¼ 100) and these were often
larger than those observed for ASH1 or SRO7 gRNA. This may
reflect a higher concentration of localized PEX3 mRNA. To
determine whether Pex3 is functional upon MS2L integration, we
examined whether PEX3INT cells grow on medium containing
oleate as a carbon source. Both wild-type and PEX3INT cells grew
on oleate unlike control pex3D cells (Fig. 3c). Thus, PEX3 mRNA
localizes to the endoplasmic reticulum, as seen previously11, and
Pex3 is functional after MS2L integration into the PEX3 locus.
Finally, we examined a mitochondria-localized mRNA, OXA1
(ref. 19), by creating an OXA1::loxP::MS2L::OXA13¢UTR strain
(OXA1INT) and expressing MS2-CP–GFP(3) (Fig. 3d). OXA1
gRNA was nonpolarized (14% of cells had gRNA at bud tip;
n ¼ 50), but colocalized/juxtaposed with Oxa1-RFP in 82% of cells
(n ¼ 50). We observed multiple granules in 25% of cells (n ¼ 100).
We observed typical tubular-punctate mitochondrial morphology
with Oxa1-RFP19. To determine whether Oxa1 is functional upon
MS2L integration, we examined OXA1INT cells for growth on
medium containing glycerol as a carbon source. Both wild-type
and OXA1INT cells grew on glycerol, unlike control oxa1D cells
(Fig. 3e). Thus, Oxa1 is functional after MS2L integration into the
OXA1 locus.
We demonstrate here a simple and efficient technique for the
tagging of genomic loci with RNA-binding protein–binding sites
that allows for the visualization of endogenous mRNA upon
MS2-CP–GFP(3) expression. m-TAG does not displace the
3¢ UTR as do other 3¢-end tagging techniques8,9 and, therefore,
allows for proper mRNA localization.
Note: Supplementary information is available on the Nature Methods website.
412 | VOL.4 NO.5 | MAY 2007 | NATURE METHODS
ACKNOWLEDGMENTS
We thank K. Bloom, P. Brennwald, J. Brickner, M. Longtine, S. Michaelis, R. Singer
and D. Stillman for the generous gifts of reagents; A. Cohen and I. Goldshtein for
technical assistance. This work was generously supported by a grant from the Kahn
Fund for Systems Biology, Weizmann Institute of Science. J.E.G. holds the Henry
Kaplan Chair in Cancer Research.
AUTHOR CONTRIBUTIONS
Experimental design was by L.H., S.A. and J.E.G.; reagent preparation by L.H.
and G.Z.; data collection, analysis, figure preparation and text by L.H. and J.E.G.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemethods/
Reprints and permissions information is available online at
http://npg.nature.com/reprintsandpermissions
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