MicroRNA-206 colocalizes with ribosome-rich regions
in both the nucleolus and cytoplasm
of rat myogenic cells
Joan C. Ritland Politz*, Fan Zhang, and Thoru Pederson
Department of Biochemistry and Molecular Pharmacology and Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605
Communicated by Masayasu Nomura, University of California, Irvine, CA, October 25, 2006 (received for review August 2, 2006)
MicroRNAs are small, ?21- to 24-nt RNAs that have been found to
regulate gene expression. miR-206 is a microRNA that is expressed
at high levels in Drosophila, zebrafish, and mouse skeletal muscle
and is thought to be involved in the attainment and/or mainte-
nance of the differentiated state. We used locked nucleic acid
probes for in situ hybridization analysis of the intracellular local-
ization of miR-206 during differentiation of rat myogenic cells. Like
most microRNAs, which are presumed to suppress translation of
target mRNAs, we found that miR-206 occupies a cytoplasmic
location in cultured myoblasts and differentiated myotubes and
that its level increases in myotubes over the course of differenti-
ation, consistent with previous findings in muscle tissue in vivo.
However, to our surprise, we also observed miR-206 to be concen-
trated in nucleoli. A probe designed to be complementary to the
precursor forms of miR-206 gave no nucleolar signal. We charac-
terized the intracellular localization of miR-206 at higher spatial
resolution and found that a substantial fraction colocalizes with
28S rRNA in both the cytoplasm and the nucleolus. miR-206 is not
concentrated in either the fibrillar centers of the nucleolus or the
dense fibrillar component, where ribosomal RNA transcription and
early processing occur, but rather is localized in the granular
component, the region of the nucleolus where final ribosome
assembly takes place. These results suggest that miR-206 may
associate both with nascent ribosomes in the nucleolus and with
exported, functional ribosomes in the cytoplasm.
microRNA ? myogenesis ? nuclear structure ? RNA localization ?
expression. In many cases they have been found to be involved
in tissue-specific translational control and are often loosely
complementary to short ‘‘seed’’ sequences in the 3? UTR of
target mRNAs (1–4). This type of control would be expected to
occur in the cytoplasm, of course, but most studies have looked
at miRNAs in whole-cell extracts and so have not defined their
intracellular locations. There is also evidence that small RNAs
operate as both positive and negative regulators of transcription,
often by altering the methylation state of chromatin (2, 5).
Until recently it has been difficult to visualize miRNAs by in
situ hybridization because their small size makes them difficult
to target specifically. However, with the advent of locked nucleic
acid (LNA) probes (6), specific hybridization to miRNAs is now
possible (7, 8). An LNA probe is a phosphodiester-bonded
oligodeoxynucleotide that contains at least one 2?-O,4?-C-
methylene-?-D-ribofuranosyl nucleotide (LNA monomer). The
probe causes the resulting DNA:RNA hybrid to resemble an
A-type RNA helix. RNA hybrids formed with LNA probes
exhibit a significantly increased Tm and ?Tm for mismatches
compared with unmodified oligodeoxynucleotides, allowing for
the specific detection of small RNA targets (6). miRNAs in-
volved in myogenesis have been identified by LNA probe in situ
hybridization studies carried out on the embryonic musculature
icroRNAs (miRNAs) are small, ?21- to 24-nt regulatory
RNAs that contribute significantly to the control of gene
of a number of species including zebrafish, Drosophila, and
mouse (8–10). miR-1 is the most well characterized of these
myogenesis-related miRNAs and is highly expressed in both
as miR-206, appear to be expressed at high levels in skeletal
muscle only (8, 12, 13).
It is thought that most miRNAs, including the aforementioned
muscle-expressed ones, are concentrated in the cytoplasm (4,
14), but this has not been investigated in detail. The embryos
investigated are not well suited for high-spatial-resolution anal-
ysis because intracellular structures are more difficult to discern
when cells overlap one another in the optical z axis. Therefore,
we decided to determine the intracellular localization of specific
miRNAs in single cultured myogenic cells using in situ hybrid-
ization followed by high-resolution imaging microscopy. We
report here an unanticipated localization pattern for the muscle-
specific miRNA, miR-206, in a rat myogenic cell line. Using
LNAs as hybridization probes, we found that miR-206 is not only
distributed throughout the cytoplasm as expected but also is
concentrated in the nucleolus. Furthermore, we demonstrate
that miR-206 partially colocalizes with 28S rRNA in the cyto-
plasm and, remarkably, also substantially colocalizes with 28S
rRNA in the granular component (GC) of the nucleolus.
To detect and localize the very small targets represented by
miRNAs, we exploited the higher specificity and hybridization
efficiency of LNA probes (6–8). Fig. 1 shows typical in situ
hybridization patterns obtained in a rat myogenic cell line with
LNAs directed against two miRNAs, miR-206 and let-7, as well
as one directed against signal recognition particle (SRP) RNA.
hybridization experiment; similar results were obtained in ex-
periments in which the probes were hybridized separately. It can
be seen that the probe for miR-206 hybridized most extensively
to nucleolar and cytoplasmic sites, with some nucleoplasmic
signal, whereas the probe for let-7 gave a different pattern with
a more uniform intracellular distribution overall. The hybrid-
ization pattern of SRP RNA with its LNA probe produced the
same intranuclear pattern as was obtained by using peptide
nucleic acid probes (SRP PNA in Fig. 1 Lower Right; also see ref.
15) and phosphodiester backbone probes (16), but hybridization
to SRP RNA was often stronger using the LNA probe (Fig. 1,
compare Upper Right and Lower Right). In both the miR-206 and
SRP LNA hybridizations, the ratio of nucleolar:nuclear signal
Author contributions: J.C.R.P. and T.P. designed research; J.C.R.P. and F.Z. performed
research; J.C.R.P., F.Z., and T.P. analyzed data; and J.C.R.P. and T.P. wrote the paper.
The authors declare no conflict of interest.
FC, fibrillar center; GC, granular component; DFC, dense fibrillar component; UBF, up-
stream binding factor.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
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December 12, 2006 ?
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varied from ?2:1 to almost 1:1, whereas in cells probed for let-7
the nucleolar signal levels never were higher than nucleoplasmic
We next assayed the levels of miR-206 during myogenic
differentiation. L6 myoblasts were grown in 2% horse serum to
induce myotube formation (17) and subjected to in situ hybrid-
ization for miR-206 at various times. The amount of signal
present in the cytoplasm, the nucleoplasm, and the nucleolus was
quantified after digital images were captured. As can be seen in
Fig. 2A, the amount of miR-206 in the cytoplasm increased
significantly during differentiation, but the amounts in the
nucleoplasm and the nucleolus remained more constant. When
a scrambled sequence LNA probe was used, a low, rather
constant level of signal was obtained over the course of differ-
entiation (Fig. 2B). The observed increase in cytoplasmic miR-
206 during myogenesis (Fig. 2A) is consistent with earlier results
showing that total cellular levels of miR-206 increase during
embryonic muscle differentiation (10) and also increase during
differentiation of another myogenic cell line (13, 18).
These results demonstrated that the LNA probe hybridized as
expected to miR-206 in the L6 myogenic cell culture system, but
we wanted to further substantiate the specificity of miR-206
probe hybridization in the nucleolus. We considered it very
unlikely that this localization pattern was due to cross-
hybridization with other nucleolar RNAs because the results of
BLAST (www.ncbi.nlm.nih.gov/BLAST and www.psb.ugent.be/
rRNA/blastrrna.html) and Ensembl (www.ensembl.org)
searches showed that the miR-206 LNA probe is not comple-
mentary to any rat ribosomal RNAs (including 28S, 18S, 5.8S, or
5S sequences) or any known expressed small nucleolar RNAs.
(These searches did locate an 11-nt complementary sequence in
a U1 snRNA pseudogene with no upstream promoter se-
quences.) However, we also tested the fidelity of the hybridiza-
tion directly with experiments in L6 myoblasts in which we varied
the stringency of the hybridization and then compared the ratio
of nucleolar to cytoplasmic signal present in digital images.
Hybridization of LNA oligos has been shown to be affected by
ionic strength and temperature in a manner similar to hybrid-
ization with standard phosphodiester backbone DNA oligos
(19), so if there were differences in hybridization fidelity be-
tween the nucleolar and cytoplasmic targets, we would expect to
see differences in the ratio under altered hybridization stringen-
cies. However, we observed no significant difference in the
nucleolus:cytoplasmic signal ratios when hybridization was car-
ried out at twice the ionic strength (ratios were 1:1 at both 4?
SSC and 2? SSC) or when a very high-stringency wash was
included (nucleolar:cytoplasmic ratios were 1:1 after washes at
1? SSC and 0.1? SSC). These results indicate that the hybrid-
ization to the detected target in each of these compartments was
equally specific. Therefore, it is highly likely that the LNA probe
is hybridizing to bona fide miR-206 sequence in the nucleolus.
Although the foregoing results indicated that the LNA probe
was indeed hybridizing to miR-206 RNA, we did not know
whether we were detecting the mature and/or the precursor
forms of this RNA (3, 14). We therefore designed a LNA probe
to the loop region of the precursor hairpin of miR-206 (see
Materials and Methods), which is expected to hybridize to both
the primary transcript (pri-miR-206) and the immediate miR-
206 precursor (pre-miR-206). This loop region of various pre-
miRNAs, including pre-miR-206, has been shown to be prefer-
entially available for hybridization in microarray assays (20). Fig.
3 shows the results of an experiment in which a probe comple-
mentary to mature miR-206, labeled with cy3 (Fig. 3A), and a
second probe complementary to the loop region of the precur-
sors of miR-206, labeled with fluorescein, (Fig. 3B) were simul-
taneously hybridized to L6 cells. The precursor-specific probe
displayed very little signal (Fig. 3B) compared with the probe for
mature miR-206 (Fig. 3A), and no specific signal was observed
in the nucleolus. However, the cytoplasmic levels for the pre-
cursor probe were slightly above those obtained by using the
scrambled control probe (Fig. 3F), especially in the perinuclear
region. We conclude that the signal we observed in the nucleolus
using the probe to the mature sequence is very likely to reflect
hybridization to the mature form of miR-206.
To confirm that the detected miR-206 signal indeed repre-
sented hybridization to RNA, we treated fixed cells with RNase
before carrying out in situ hybridization with the miR-206 probe.
Only low level of signal was observed in these cases (Fig. 3 G and
H), indicating that the probe was hybridizing to RNA in both the
nucleus and the cytoplasm. When cells were treated with DNase
distribution remained unchanged (data not shown).
We next examined the nucleolar localization of miR-206 at
higher spatial resolution. The nucleolus has been classically
defined as having three components by ultrastructural criteria
(21): the fibrillar centers (FCs), where the rDNA genes are
located; the dense fibrillar component (DFC), which immedi-
ately surrounds the FCs and into which the prerRNA nascent
transcripts extend and initial processing events take place; and
miR-206 let-7 SRP LNAmiR-206 let-7 SRP LNA
RNA. (Upper) Distribution of signal after in situ hybridization using LNA
probes to miR-206, let-7, and SRP RNA. Images are not scaled to the same
intensity ranges. All probes were cy3-labeled except let-7, which was fluores-
cein-labeled; miR-206 and let-7 patterns are shown after a dual hybridization
experiment. (Lower) The corresponding phase images for miR-206 and let-7.
(Lower Right) The hybridization pattern to SRP RNA using a peptide nucleic
acid probe. PNA, peptide nucleic acid. Arrows point to the nucleolus. Each
image is 35 ?m wide.
In situ hybridization pattern of miR-206 compared with let-7 and SRP
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
levels of miR-206 in L6 nucleoli, nucleoplasm, and cytoplasm during myogen-
esis. (B) Same as A, but a scrambled control probe was used for hybridization.
Similarly sized regions in each compartment were circled, and the average
intensity per pixel was recorded. The averages of these measurements in
multiple cells are shown here. It should be noted that this experiment has a
low signal-to-noise ratio compared with the other experiments presented in
this article because it was necessary to use confluent cultures to induce
differentiation. In situ hybridization signal is substantially reduced in conflu-
ent cell cultures because of permeability issues. Bars indicate standard errors.
www.pnas.org?cgi?doi?10.1073?pnas.0609466103Politz et al.
the GC, where additional prerRNA processing and ribosome
assembly take place. In addition to the classically identified
ribosomal components, other proteins and RNAs that are not
known to be linked to the ribosome assembly pathway have been
detected in the nucleolus, including SRP RNA (15, 16), nu-
cleostemin (22), telomerase components (23–25), and various
cell cycle-related proteins (26, 27).
Fibrillarin, a protein complexed with small nucleolar RNAs
involved in rRNA processing (28), is commonly used to demar-
cate the DFC. Fig. 4 shows the typical results of experiments in
which L6 myoblasts were either transfected with plasmids coding
for GFP-fibrillarin (Fig. 4 A–E and K) or immunostained with
antibodies to fibrillarin (Fig. 4 F–J and L) and then subjected to
in situ hybridization to detect miR-206. Fig. 4A shows the
hybridization pattern for miR-206 after cells were transfected
with GFP-fibrillarin and then fixed (Fig. 4B), and Fig. 4F shows
the hybridization pattern for miR-206 after cells were fixed and
then subjected to immunostaining for fibrillarin (Fig. 4G).
Comparison of the miR-206 hybridization pattern in Fig. 4 A and
F shows that, after cells have been immunostained and then
subjected to in situ hybridization, much of the cytoplasmic and
nucleoplasmic miR-206 signal is lost, whereas the amount of
nucleolar miR-206 signal stays the same or slightly increases as
compared with results from the in situ hybridization after
transfection with the GFP-fibrillarin plasmid. We believe that
this is because the small miR-206 target molecules are washed
out of the cytoplasm and nucleoplasm during the immunostain-
ing procedure, because similar experiments with SRP LNA
probes did not show reduced detection of the (15-fold) longer
SRP RNA (data not shown).
A B C
D E F
to premiR-206, and hybridization is RNase-sensitive. Dual color in situ hybrid-
ization was performed by using a cy3-labeled LNA probe to miR-206 (A) and
a fluorescein-labeled LNA probe to premiR-206 (B). (C) Phase contrast image.
Red arrows point to the nucleoli. (D and E) Control using cy3-labeled LNA
miR-206 probe alone. (D) Red channel. (E) Green channel. This image shows a
nucleus where the nucleolar:nucleoplasmic ratio of miR-206 is ?1:1. (F) Cy3-
in situ hybridization with cy3-labeled miR-206 LNA probe. (H) Cells were
treated with a mixture of three ribonucleases (see Materials and Methods)
before in situ hybridization with cy3-labeled miR-206 LNA probe. G and H are
scaled the same. All images are 35 ?m wide.
Mature miR-206 probe hybridizes in a different pattern than a probe
miR-206 fibrillarin-GFP (DFC)
C D E
miR-206 fibrillarin Ab (DFC)
H I J
109 119 129
139 149 159
transfected with a plasmid encoding GFP-fibrillarin (A–E) or subjected to
immunostaining for fibrillarin (F–J) followed by in situ hybridization for
A and B show raw images of miR-206 and fibrillarin-GFP signal. C and E show
the corresponding deconvolved nucleoli. D shows a pseudocolor image of C
and E overlap where miR-206 is red and fibrillarin is green. Similarly, F and G
show raw images of miR-206 and fibrillarin signal after antibody staining and
in situ hybridization, H and J show the corresponding deconvolved nucleoli,
and I shows a pseudocolor image of H and J overlap. Yellow indicates areas of
minimal overlap between fibrillarin and miR-206. (K) Linescan showing inten-
sity of both miR-206 (red) and fibrillarin-GFP (green) along the line shown in
G), 5.7 ?m wide (C–E), and 6.8 ?m wide (H–L).
miR-206 is not concentrated in nucleolar DFC. L6 myoblasts were
Politz et al.PNAS ?
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Images obtained after iterative deconvolution analysis (22, 29)
of the intranucleolar distribution of miR-206 with respect to
GFP-fibrillarin distribution are shown in Fig. 4 C–E, and simi-
larly the results with fibrillarin antibody are shown in Fig. 4 H–J.
Both detection methods revealed similar nucleolar distribution
patterns, and it can be seen that there was very little overlap
miR-206 (red) and fibrillarin (green) in either case. Fig. 4 K and
L shows intensity linescans across the regions in Fig. 4 D and I,
revealing the extent of miR-206 overlap with GFP-fibrillarin and
fibrillarin antibody, respectively. These results show that miR-
206 is not appreciably present in the DFC of the nucleolus.
We then looked to see whether miR-206 was present in the
FCs, using as a fiduciary marker the upstream binding factor
(UBF), a protein that specifically binds the rDNA across the
entire rDNA repeat (30). Fig. 5 A–H shows two examples of
nucleoli stained with UBF antibody (green) and hybridized with
the miR-206 LNA probe (red), with the intensity linescans for
the color overlays in Fig. 5 B and E shown in Fig. 5 G and H,
We next examined the localization of miR-206 within the GC
by comparing its hybridization pattern to the distribution of
nucleostemin within this compartment (Fig. 5 I–P). Our earlier
work with nucleostemin has shown that this protein was a good
marker for this region (22). Fig. 5 I–K and L–N show decon-
volved midplanes from nucleoli of two different cells. Fig. 5 O
and P shows intensity distributions along the lines depicted in the
pseudocolored images in Fig. 5 J and M, respectively, and
represent typical results. Linescans through some nucleolar
regions showed virtually complete overlap (Fig. 5P) whereas
others showed a much lesser degree of overlap (Fig. 5O),
indicating that miR-206 partially, but not completely, overlaps
with the regions of the GC occupied by nucleostemin. Taken
together, these nucleolar mapping results demonstrate that the
miR-206 is primarily localized to the GC.
To learn more about the possible associations of miR-206 in
the intracellular localization of miR-206 to that of 28S rRNA.
We performed double in situ hybridization experiments to detect
miR-206 and 28S rRNA and found that miR-206 colocalizes to
a significant extent with 28S rRNA in both the nucleolus and the
enlarged images of nucleoli, with accompanying linescans of the
combined color-coded images. The yellow in the pseudocolored
images (Fig. 6 B, D, H, and J), as well as the presence of
overlapping maxima and minima in the linescans (even though
not always the same intensity; Fig. 6 E, F, K, and L), demonstrate
the substantial colocalization of these two probes. A significant
overlap between miR-206 and 28S rRNA in both the nucleolus
and the cytoplasm was observed in all cells, and in some regions
the extent of spatial overlap was almost complete (e.g., Fig. 6K).
These results demonstrate that miR-206 extensively colocalizes
with 28S rRNA in both the cytoplasm and the GC of the
We have presented evidence that a miRNA involved in muscle
development concentrates not only in the cytoplasm, but also in
the nucleolus of cultured rat myogenic cells. Although nucleolar
localization has not been reported for any other miRNA, it is too
soon to know whether others visit the nucleolus because very
little high-resolution work has been done in this relatively new
field of miRNA cell biology. Indeed, while this manuscript was
in preparation, endogenous siRNAs were reported to localize to
the nucleolus in Arabidopsis (31), and there is evidence that
microinjected siRNAs visit and sometimes concentrate in the
We found that miR-206 concentrates in the nucleolar GC, and
not in the other two major compartments of the nucleolus, the
FCs or the DFC. The GC is the region of the nucleolus where
204060 80 100120140
GC. L6 myoblasts were subjected to immunostaining for UBF (to mark FCs,
A–H) or nucleostemin (to mark the GC, I–P) followed by in situ hybridization
to miR-206, and image stacks were captured and subjected to 3D deconvolu-
tion. Enlarged images of deconvolved nucleoli show miR-206 (A and D) and
show miR-206 (red) and UBF (green) overlap (yellow). Linescan in G corre-
enlarged images of deconvolved nucleoli show miR-206 (I and L) and corre-
sponding nucleostemin (K and N) distribution, and pseudocolored images (J
and M) show miR-206 (red) and nucleostemin (green) overlap (yellow). Lines-
can in O corresponds to the line in J, and linescan in P corresponds to the line
in N. Images are 9.9 ?m wide (A–C), 5.9 ?m wide (D–F), 7 ?m wide (I–K), and
7.3 ?m wide (L–N).
www.pnas.org?cgi?doi?10.1073?pnas.0609466103Politz et al.
and where a number of nonribosomal components, including
nucleostemin, also concentrate (22, 33). Dual in situ hybridiza-
tion in the present study showed that a substantial fraction of
nucleolar miR-206 colocalizes with the 28S rRNA in the GC.
Furthermore, a substantial portion of cytoplasmic miR-206 was
observed to be colocalized with 28S rRNA. This cytoplasmic
colocalization is consistent with the current hypothesis that
miRNAs control gene expression by interfering with translation
(1, 2, 4), and one recent study has suggested that at least some
miRNAs may associate with polysomes (34) and thus directly
interfere with translation at the elongation stage. Therefore,
although an association of miR-206 with polysomes has yet to be
examined in cell fractionation studies, our finding that miR-206
colocalizes with 28s rRNA in the cytoplasm suggests that it may
be regulating target mRNAs in this way.
However, our discovery that the miR-206 is also localized in the
nucleolus finds no obvious explanation in any previous or contem-
above, a recent study showed that endogenous siRNAs, which are
closely related to miRNAs, localize to the nucleolus in Arabidopsis
(31). It was proposed that siRNAs go to the nucleolus for modifi-
cation and assembly, similar to other small RNAs that visit the
nucleolus to be modified and/or assemble into RNP complexes (16,
35). At least some mammalian miRNAs are subject to A-to-I
editing by adenosine deaminases that act on RNA (36, 37), and
RNA editing enzymes have been found associated with the nucle-
olus in cultured mammalian cells (38). It has been proposed that
adenosine deaminases that act on RNA compete with Drosha and
thus regulate the processing of miRNAs in the nucleus (36, 39). It
has also been shown that the 3? terminal nucleotides of Arabidopsis
miRNAs are subject to ribose-2?-O-methylation (40), but there are
no reports so far of this modification in human miRNAs, much less
any reports describing the intracellular compartment at which this
Although the above explanation for the nucleolar localization
of miR-206 is reasonable, it does not explain why a substantial
portion of miR-206 colocalizes with 28S rRNA in the nucleolus.
It seems unlikely that miR-206 is involved in regulating tran-
the known sites of these genes in the nucleolus, the FCs. This
possibility was not trivial, because recent studies have implicated
small noncoding RNAs, produced as intergenic transcripts
within the rDNA repeats, in the transcription regulation of these
genes (41). Furthermore, it is not likely that miR-206 is directly
targeting rRNA transcripts in the way that miRNAs are pre-
dicted to target messenger RNAs, because we found no 7- or 8-nt
sequence elements complementary to the 8-nt seed sequence of
miR-206 in rat 28S, 5.8S, or 5S rRNAs. (The seed sequence is the
region of miRNAs that is thought to hybridize most strongly to
the target mRNA.) We did find one 18S site that contained 7/8
complementary nucleotides to the miR-206 seed sequence;
rest of miR-206. Nevertheless, we cannot rule out the possibility
that miR-206 hybridizes with different affinity to rRNA or
associates with certain nascent ribosomal subunits through in-
teractions other than hybridization to the rRNA.
A final intriguing possibility is that miR-206 could be targeting
messenger RNAs that transit through the nucleolus, examples of
which have been reported (42, 43), and perhaps miR-206 even
moves with these messages to the cytoplasm. Certain proteins
have been proposed to associate with messenger RNAs in the
nucleolus and then move as a messenger ribonucleoprotein
complex to the cytoplasm for translation (44), and it is possible
that certain miRNAs may do the same. Of course, an experi-
mental demonstration of the functional significance of the
nucleolar concentration of miR-206 awaits further investigation.
Materials and Methods
In Situ Hybridization Probes. LNA hybridization probes comple-
mentary to rat mature miR-206 and let-7a were purchased from
Exiqon (Vedbaek, Denmark) with both 5? and 3? aminohexyl
groups and labeled with cy3 as described (45). The miRNA
sequences are given at http://microrna.sanger.ac.uk (46, 47). An
LNA probe complementary to SRP RNA was designed to target
nucleotides 228–248. Its sequence was 5?-Agg cgc gAt ccc Act
acT gat-3?, where capital letters indicate the presence of a locked
nucleotide. An LNA probe complementary to the loop region of
rat premiR-206 (http://microrna.sanger.ac.uk) (46, 47) was de-
signed and had the sequence 5?-ttCcAtaGcGcaGtGaTatCta-3?.
A control nonhybridizing LNA was designed with the sequence
5?-acgtgaCaCgttcgGagAatt-3?, as suggested by Faba Neumann
and Jan Ellenberg (personal communication). These probes
were also synthesized by Exiqon and labeled with either fluo-
probes (48) and the SRP peptide nucleic acid probe (15) were as
Cell Growth and Fixation. L6 cells (from cultures that had been
passaged ?10 times) were plated on 25-mm coverslips in six-well
tissue culture dishes in DMEM containing 10% FBS. Cells were
310 360410 460
300 320 340
the cytoplasm. A–F and G–L depict results from two different cells. A and G
show the miR-206 signal in a deconvolved mid-plane of the cell, and C and I
show the 28S rRNA signal in the same plane. B and H are pseudocolored
images combined to reveal overlap. D and J show enlarged images of the
nucleoli. E and K are intensity linescans along the white line in D and J,
respectively. F and L are intensity linescans along the white line in B and H,
respectively. Images are 35 ?m wide (A–C and G–I), 5.8 ?m wide (D), and 4.1
?m wide (J).
miR-206 partially colocalizes with 28S rRNA in the nucleolar GC and
Politz et al. PNAS ?
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cultured overnight at 37°C in 5% CO2, reaching a confluency of
?50–60%. The coverslips were rinsed once with PBS, fixed in
4% formaldehyde/5 mM MgCl2in PBS at 20–21°C for 15 min,
and then stored in 70% ethanol at 4°C at least overnight and for
up to 1 month. Alternatively, after overnight growth, the me-
the cells were allowed to differentiate and fuse into myotubes
over 5–7 days and then fixed and stored as above.
In Situ Hybridization and Immunostaining. Fixed cells were rehy-
drated in 5 mM MgCl2in PBS and then prehybridized in 40%
formamide in 2? SSC (1? SSC ? 0.15 M NaC1/15 mM sodium
citrate, pH 7.0) for 10 min each at 20–21°C. Twenty nanograms
of probe and 5 ?g each of Escherichia coli tRNA and salmon
sperm DNA (per coverslip) were heated at 94–95°C in 10 ?l of
80% formamide in diethylpyrocarbonate-treated water for 3–5
min, removed from the heating block, and immediately mixed
with an equal volume of hybridization buffer (2 mg/ml BSA/20%
dextran sulfate in 4? SSC) at 20–21°C. Twenty-microliter ali-
quots were placed on parafilm stretched on a glass plate,
hybridization was allowed to proceed at 37°C for 3 h. Coverslips
were then washed for 10 min at 37°C with 40% formamide in 2?
SSC, then twice for 30 min each at 37°C with 40% formamide in
1? SSC, and then at 20–21°C for 15 min once with 1? SSC and
once for 15 min with 5 mM MgCl2in PBS. Coverslips were then
mounted in Prolong Gold Antifade (Invitrogen, Carlsbad, CA)
and allowed to dry overnight in the dark at 20–21°C before
observation and imaging. Immunostaining for UBF and fibril-
larin and transfection of GFP-fibrillarin were performed as
described (15), and after refixation the cells were subjected to in
situ hybridization as described above. For RNase experiments,
fixed cells were rehydrated, washed in RNase buffer (150 mM
NaCl/1.5 mM MgCl2/10 mM Tris?HCl, pH 7.5), and then incu-
bated with RNase buffer alone or with RNases T2 (20 units), T1
(20 units), and A (0.5 units) in 20 ?l per coverslip for 1 h at 37°C,
followed by two 10-min washes with 5 mM MgCl2 in PBS at
20–21°C followed by the prehybridization wash and in situ
hybridization as described above.
Microscopy and Image Processing. Microscopy, image processing,
and deconvolution analysis were as described (22, 49). A long-
pass TRITC filter cube (Leica M2) was used to detect red
fluorescence, and a band-pass ‘‘GFP’’ filter cube (Chroma
41017) was used to detect green fluorescence. All dual hybrid-
ization experiments were designed using the appropriate con-
trols to ascertain that there was no bleed-through between the
We are grateful to Kevin Fogarty and Larry Lifshitz (Biomedical
Imaging Facility, University of Massachusetts Medical School) for help
with deconvolution analysis and Faba Neumann and Jan Ellenberg
(European Molecular Biology Laboratory, Heidelberg, Germany) for
suggesting the sequence for the nonhybridizing control probe. This work
was supported by National Institutes of Health Grant GM-60551 and
National Science Foundation Grant MCB-0445841.
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www.pnas.org?cgi?doi?10.1073?pnas.0609466103Politz et al.
CELL BIOLOGY. For the article ‘‘MicroRNA-206 colocalizes with
ribosome-rich regions in both the nucleolus and cytoplasm of rat
myogenic cells,’’ by Joan C. Ritland Politz, Fan Zhang, and
of Proc Natl Acad Sci USA (103:18957–18962; first published
November 29, 2006; 10.1073?pnas.0609466103), the authors
note that in Fig. 5, A and C were transposed. The corrected
figure and its legend appear below. This error does not affect the
conclusions of the article.
GC. L6 myoblasts were subjected to immunostaining for UBF (to mark FCs, A–H)
or nucleostemin (to mark the GC, I–P) followed by in situ hybridization to
miR-206, and image stacks were captured and subjected to 3D deconvolution.
Enlarged images of deconvolved nucleoli show miR-206 (A and D) and corre-
sponding UBF (C and F) distribution, and pseudocolored images (B and E) show
(K and N) distribution, and pseudocolored images (J and M) show miR-206 (red)
5.9 ?m wide (D–F), 7 ?m wide (I–K), and 7.3 ?m wide (L–N).
miR-206 is not concentrated in nucleolar FCs but is concentrated in the
January 9, 2007 ?
vol. 104 ?
MICROBIOLOGY. For the article ‘‘A deletion defining a common
Asian lineage of Mycobacterium tuberculosis associates with
immune subversion,’’ by Sandra M. Newton, Rebecca J. Smith,
Katalin A. Wilkinson, Mark P. Nicol, Natalie J. Garton, Karl J.
Staples, Graham R. Stewart, John R. Wain, Adrian R. Martin-
eau, Sarah Fandrich, Timothy Smallie, Brian Foxwell, Ahmed
Al-Obaidi, Jamila Shafi, Kumar Rajakumar, Beate Kampmann,
Peter W. Andrew, Loems Ziegler-Heitbrock, Michael R. Barer,
2006, of Proc Natl Acad Sci USA (103:15594–15598; first pub-
lished October 6, 2006; 10.1073?pnas.0604283103), the authors
note that the y axes of Fig. 4 B and D were labeled incorrectly.
The corrected figure and its legend appear below. These errors
do not affect the conclusions of the article.
of deleted genes into CH on IL-10 secretion and promoter activity. (A) The 24-h
in cultures stimulated by CH and CAS2 such that the ratio was ?1 log higher for
both strains when compared with H37Rv or CDC1551. (B) Three-day MDMs
infected with the pAdT-IL-10.wt-luc adenovirus were treated with heat-killed
MTB samples. LPS (100 ng/ml) was used as a positive control. Data were normal-
experiments. IL-10 promoter activity was greater in cultures stimulated with
heat-killed CH than in those stimulated with H37Rv (P ? 0.05). (C) MDMs from
seven donors were cocultured with strains. Strain CH induced significantly more
that of H37Rv in all donors (P ? 0.016 by comparison with CH). (D) Three-day
MDMs infected with the pAdT-IL-10.wt-luc adenovirus were treated with heat-
killed MTB samples. Data were normalized to protein concentration and are
was greater in cultures stimulated with heat-killed CH than in those stimulated
with CH::Rv1519 (P ? 0.05).
January 9, 2007 ?
vol. 104 ?
no. 2 ?