MicroRNAs with a nucleolar location
JOAN C. RITLAND POLITZ,1,2,3ERIC M. HOGAN,1,2and THORU PEDERSON1,2
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
2Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the
microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here
we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs
can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced
nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and
occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs
using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor
form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites
within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a
portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not
show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as
modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have
recently been ascribed to the granular component of the nucleolus.
Keywords: microRNAs; nucleolus; nucleus; muscle; nuclear RNAs; in situ nucleic acid hybridization
MicroRNAs (miRNA) have been known and studied for a
number of years now, but their spatial disposition within
the cell has sometimes been difficult to determine. Many
miRNAs are transcribed as large primary transcripts,
sometimes containing more than one embedded miRNA
element, which are commonly processed into precursor
‘‘hairpin’’ miRNAs by Drosha/DGCR8 in the nucleus (Zeng
et al. 2005; Yeom et al. 2006; Carthew and Sontheimer
2009). This processing can occur cotranscriptionally
(Morlando et al. 2008; Pawlicki and Steitz 2008) and is
subject to regulation (Thomson et al. 2006; Lee et al. 2008).
For example, lin-28 can bind to pri-let-7 at some stages
during neuronal development and inhibit its processing to
the mature form (Wulczyn et al. 2007; Newman et al.
2008). Drosha-generated precursors are transported to the
cytoplasm where Dicer further processes them into small
double-stranded RNAs that interact with Argonaute pro-
teins (Matranga and Zamore 2007; Carthew and Sontheimer
2009). Like Drosha processing, this step is also subject to
regulation (Lee et al. 2008). Additionally, some miRNA
transcripts are also subject to A/I editing (Luciano et al.
2004; Blow et al. 2006; Habig et al. 2007; Kawahara et al.
2008), and such editing can interfere with processing (Yang
et al. 2006; Kawahara et al. 2007). miRNAs that have been
diced and assembled into an Argonaute complex then
interact via short ‘‘seed’’ sequences with mRNAs in the
cytoplasm to regulate translation (Du and Zamore 2005;
Filipowicz et al. 2008), and in some cases, miRNAs have
been isolated in polysomal complexes (Kim et al. 2004; see
Wang et al. 2008).
There is also mounting evidence that small RNAs, in-
cluding miRNAs, play important nuclear roles and that
some miRNAs, once exported to the cytoplasm, may revisit
the nucleus (Hwang et al. 2007; Guang et al. 2008; Kim
et al. 2008; Marcon et al. 2008; Ohrt et al. 2008; Place et al.
2008). The nuclear site(s) and the functional significance of
these returning miRNAs are not known.
During a study of miRNAs expressed during muscle
cell differentiation (Politz et al. 2006), we observed that
3Present address: Division of Basic Sciences, Fred Hutchinson Cancer
Research Center, Seattle, WA 98109, USA.
Reprint requests to: Joan C. Ritland Politz, Division of Basic Sciences,
Mailstop A3-025, Fred Hutchinson Cancer Research Center, Seattle, WA
98109, USA; e-mail: email@example.com; fax: (206) 667-5939.
Article published online ahead of print. Article and publication date are
RNA (2009), 15:1705–1715. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2009 RNA Society.
miR-206, a microRNA implicated in muscle cell differen-
tiation and maintenance (Sempere et al. 2004; Kim et al.
2006; McCarthy 2008; van Rooij et al. 2008), is concen-
trated in the nucleoli of a rat myoblast cell line. We decided
to follow up on this observation to determine whether this
nucleolar localization was specific to miR-206 or muscle-
specific miRNAs or whether many miRNAs might visit the
nucleolus. The nucleolus is a tripartite structure in terms of
known functions and corresponding fiduciary molecular
markers (Huang 2002; Raska et al. 2006; Boisvert et al.
2007). Preribosomal RNA is transcribed at so-called fibril-
lar centers, and initially processed in a surrounding domain
known as the nucleolar dense fibrillar component (DFC),
where fibrillarin and GAR1, among other proteins, are
complexed with small nucleolar RNAs (snoRNAs) with the
resulting snoRNPs modifying rRNA at multiple sites (Ochs
et al. 1985; Matera et al. 1994, 2007; Pogacic et al. 2000;
Gerbi et al. 2003). A third nucleolar domain, the granular
component (GC), harbors the late stages of ribosome
biosynthesis, where some rRNA modification still occurs
and snoRNPs also accumulate. Although the nucleolus has
classically been viewed solely as the site of ribosome
biosynthesis, more recently it has been found that other
proteins and RNAs also localize therein, including com-
ponents involved in cell cycle control (Pederson 1998;
Visintin and Amon 2000; Raska et al. 2006; Ma and
Pederson 2008b; Pederson and Tsai 2009). We earlier
determined that miR-206 colocalizes with 28S rRNA in
the GC, where some of the newly identified proteins with
nonribosomal functions also concentrate (Karayan et al.
2001; Politz et al. 2002, 2005; Ma and Pederson 2008a).
We have expanded our studies and now report that a
number of miRNAs associate with the nucleolus in rat
myoblasts. Some of these are significantly concentrated in
the nucleolus relative to their prevalence in the nucleo-
plasm and/or the cytoplasm. We have also defined the
intranucleolar localization of three of the most highly
nucleolus-concentrated miRNAs and find that they are
clustered, like miR-206, in the GC, raising the possibility
that they may be involved in common functions at these
To systematically determine which miRNAs might be
present in the nucleolus, nuclei were isolated from L6 rat
myoblasts using a protocol optimized specifically for this
cell type (Mellon and Bhorjee 1982). We found that this
method exactly replicated the high nuclear purity initially
reported. The cytoplasmic fraction was saved, and the
nuclear fraction was subjected to a nucleolus:nucleoplasm
separation protocol (Maggio et al. 1963; Muramatsu et al.
1963) modified for cultured mammalian cells by our lab-
oratory (Bhorjee and Pederson 1972, 1973). Total RNA was
then isolated from each fraction and analyzed by micro-
Because mature miRNAs are so small, their detection by
hybridization represents a technical challenge. Accordingly,
we employed a system that uses locked nucleic acid (LNA)
probes, which are phosphodiester backbone oligodeoxy-
nucleotides containing at one or more internal sites a
bicyclonucleoside that has a methylene group bonded to
the ribose 29-OH and C94. This locks the ribose into the
C39-endo pucker and confers exceptional stability when the
LNA is hybridized to a complementary target in either
microarray assays or during in situ hybridization (Thomsen
et al. 2005; Ason et al. 2006; Castoldi et al. 2008). Each LNA
monomer raises the Tmof the probe:RNA hybrid by 2°C–
8°C, and the specificity of the hybridization increases such
that single nucleotide differences between miRNAs can
often be distinguished. We used the LNA-based microRNA
profiling analysis miChip (Castoldi et al. 2008) to screen for
the full complement of rat miRNAs recorded in miRBase
(version 9.2, Wellcome Trust Sanger Institute). The LNAs
on these microarray chips are designed to detect mature,
primary, and precursor forms of miRNAs, which allowed
us to screen for nucleolar miRNAs that concentrate in the
nucleolus in any form.
The purity of each cellular fraction was assayed by
microscopy during purification (Supplemental Fig. 1) and
was subsequently corroborated by the relative abundance of
FIGURE 1. Purity of nucleolar fraction. (A) Normalized log signal in
nucleolar, nucleoplasmic, and cytoplasmic fractions from L6 rat
myoblasts to human U6 and snoRNA probes (provided as controls
on Exiqon miRCURY chips). Signal (Hy3) was normalized using total
RNA (Hy5), which was an equimolar mixture of nucleoplasmic,
nucleolar, and cytoplasmic RNA. The sequence of human and rat U6
are identical at 104/106 nt. Human snoRNAs have varying homology
with rat snoRNAs, but both homologous and nonhomologous human
snoRNAs are shown to demonstrate specificity and purity of the rat
cell fractions used in this study. Cross-species homology is as follows:
hsa_SNORD6, 4-nt difference; hsa_SNORD4A, expression not veri-
fied in rat; hsa_SNORD3@, 3-nt difference; hsa_SNORD2, 4-nt
difference; hsa_SNORD15A, 18-nt difference; hsa_SNORD14B, not
identified in rat; hsa_SNORD13, 19-nt difference; and hsa_SNORD12,
9-nt difference. (B) RT-qPCR of rat Y1 RNA and rat snoRNA E2
showing relative quantities (RQ) in nucleolar, nucleoplasmic, and
cytoplasmic fractions from L6 rat myoblasts.
Politz et al.
RNA, Vol. 15, No. 9
compartment-specific RNAs as detected by both the micro-
array and RT-qPCR (Fig. 1). Known snoRNAs were present
at high levels in the nucleolus-enriched fractions and at
much lower levels in nucleoplasm- and cytoplasm-enriched
fractions. The U6 small spliceosomal RNA was present at
similar levels in the nucleoplasm, where it functions, and
the nucleolus, where it traffics for ribose modification
(Fig. 1A; Ganot et al. 1999). Since most nucleoplasm-
concentrated small RNAs traffic through the nucleolus, we
were unable to test a marker completely specific for the
nucleoplasm. The fractionation was further substantiated
by RT-qPCR analysis of marker RNAs. Thus, a known
snoRNA, E2 (Mishra and Eliceiri 1997), was present at
highest levels in the nucleolus-enriched fraction, while a Ro
small RNA family member, Y1 RNA, was detected at
highest levels in the cytoplasmic fraction, as expected for
this known cytoplasm-concentrated RNA species (Fig. 1B;
Hendrick et al. 1981).
The microarray results revealed that at least one third
of the 280 rat miRNAs probed on the microarray were
expressed in L6 myoblasts, and surprisingly, most of these
showed a significant presence in the nucleolus (Supple-
mental Table 1). Indeed, one third of the detected miRNAs
exhibited nucleolar levels at least as high as those observed
in the cytoplasm (Supplemental Table 2). The majority of
these miRNAs are not known to be involved specifically in
muscle differentiation, so these results suggest that nucle-
olar localization is not unique to muscle-specific miRNAs
but rather is a more general phenomenon. As expected,
many miRNAs showed cytoplasmic levels higher than the
nucleoplasmic and nucleolar signals, and not all expressed
miRNAs were detectable in the nucleolus. About 7% of the
miRNAs showed nucleolar levels far below their respective
levels in the nucleoplasm and/or the cytoplasm, thus
indicating that presence in the nucleolus is not an oblig-
atory step in miRNA biogenesis and pointing to the
specificity of this finding.
Five miRNAs, miR-340-5p, miR-351, miR-494, miR-664,
and let-7e, were significantly concentrated (two- to eightfold)
in the nucleolus compared with the nucleoplasm and/or the
cytoplasm (Fig. 2A; Supplemental Table 1). None of these
are muscle-specific miRNAs (i.e., miR-1, miR-133, miR-
206, and perhaps miR-95, miR-128a, miR-499) (Lee et al.
2008; McCarthy 2008), although one of these miRNAs has
been linked to muscle differentiation, viz. miR-494 is
expressed at elevated levels in some muscle diseases (van
Rooij et al. 2006; Eisenberg et al. 2007). miR-340-5p and
miR-351 were originally identified as part of a large miRNA
group in neurons (Kim et al. 2004). miR-351, along with
many other stress response miRNAs, has been shown to be
up-regulated in cardiohypertrophy (van Rooij et al. 2006).
Neither let-7e nor miR-664 has reported links to muscle
Three of these five miRNAs, miR-351, miR-494, and
miR-664, were chosen for further characterization (Fig.
2B). Let-7e was not pursued because although its nucleolar
levels were significantly higher than its cytoplasmic levels,
its even higher nucleoplasmic levels (see Supplemental
Tables 1, 2) might have complicated analysis. miR-340-5p
was also not pursued further because its sequence is not
annotated in the rat genome to date (although it is in other
species). We labeled LNA in situ hybridization probes to
miR-351, miR-494, and miR-664 as well as the muscle-
specific miR-206 and its family member miR-1, which is
found at elevated levels in both cardiac and skeletal muscle
(Kim et al. 2006; Rosenberg et al. 2006; McCarthy 2008;
Sweetman et al. 2008). Both miR-1 and miR-206 were
detected at high levels in the cytoplasm in the microarray
analysis as expected. But somewhat surprisingly in view of
our earlier results (Politz et al. 2006), the microarray assay
showed nucleolar/nucleoplasmic ratios closer to one for
both of these miRNAs (Fig. 2B). To further explore this
observation, we made probes to an additional four miRNAs
that also showed nucleolar/nucleoplasmic levels close to
1, miR-199a-3p, miR-21, miR-125a-5p, and let-7a (Fig.
2B), and additionally, exhibited high affinity LNA hybrid-
ization during the microarray profiling (data not shown).
We were also interested in miR-199a-3p and miR-21
because they had been shown to increase during cardiac
hypertrophy (van Rooij et al. 2006; Thum et al. 2008) and
Duchenne muscular dystrophy (Eisenberg et al. 2007).
miR-125a-5p and let-7a are both expressed ubiquitously
(Lee et al. 2008).
LNA probes to these various miRNAs (Fig. 2b) were hy-
bridized to fixed rat myoblasts using previously established
hybridization conditions (Politz et al. 2006). Figure 3 shows
that all three of the miRNAs indicated to be nucleolus-
concentrated in the microarray assays also showed nucle-
olar concentration as detected by in situ hybridization.
Both miR-351 and miR-494 were present at higher levels in
the nucleolus compared with the nucleoplasm as deter-
mined by in situ hybridization, correlating well with the
FIGURE 2. Microarray analysis of nucleolar, nucleoplasmic, and cy-
toplasmic subcellular fractions from L6 rat myoblasts. miCURY LNA
miChips were used for analysis. (A) Heat map showing relative levels
of five significantly nucleolus-concentrated miRNAs. NO indicates
nucleolus; NU, nucleus; and CY, cytoplasm. (B) Normalized Hy3/Hy5
ratios of selected miRNAs showing relative signal detected in each
fraction. Hy3 indicates Hy3-labeled RNA from single fraction; Hy5,
Hy5-labeled pooled RNA used as normalization control, containing
equal amounts of nucleolar, nucleoplasmic, and cytoplasmic RNA.
miRNAs in the nucleolus
microarray findings. Indeed, the nucleolar level of miR-351
determined by in situ hybridization was higher than that in
either the cytoplasm or the nucleoplasm, as in the micro-
array analysis. Moreover, miR-494 nucleolar levels were
higher than in the nucleoplasm but not higher than in the
cytoplasm, also as observed in the microarray data. We also
detected high levels of miR-664 in the nucleolus using in
situ hybridization, but miR-664 did not always appear
more concentrated in the nucleolus than in the nucleoplasm.
As mentioned, we had earlier observed (Politz et al.
2006), and show here for comparison (Fig. 3), that miR-
206 was concentrated in the nucleolus compared with the
nucleoplasm in about half the cells in any given population,
with an additional, more substantial presence in the cy-
toplasm. In the present study miR-1, like miR-206, showed
concentrated nucleolar signal in some but not all cells.
Figure 3 shows an example of a cell with strong miR-1
nucleolar hybridization. Such inter-cell differences during
in situ hybridization can reflect true differences in nucleolar
concentration between cells or, alternatively, be due to
variations in probe accessibility among cells. However, if
miR-1 and miR-206 were actually concentrated in all
nucleoli, we would have expected to see high nucleolus/
nucleoplasm ratios of miR-206 and miR-1 in the micro-
array profiles (where miRNAs are deproteinized and
uniformly available for hybridization). Instead, the micro-
array data showed statistically similar levels of miR-206 and
miR-1 in both the nucleolus and nucleoplasm. Taken to-
gether, these results suggest that these muscle cell-expressed
miRNAs are actually concentrated in some nucleoli and not
in others in a given rat myoblast population. The overall
moderate levels of these miRNAs in the nucleolus-enriched
biochemical fraction used for the microarray analysis thus
more likely reflect the fact that the RNAs are derived from a
mixed population of cells with higher versus lower nucle-
olar levels of miR-206 and miR-1.
The remainder of the miRNAs selected for further
characterization did not show nucleolar concentration in
the in situ hybridization experiments, although it should be
stressed that, as in the microarray results, this does not
mean there was no nucleolar presence at all. miR-199a-3p
and miR-125a-5p appeared more concentrated in the
nucleoplasm and cytoplasm compared with the nucleolus;
their hybridization pattern more closely resembled that of
let-7a, a microRNA that earlier in situ hybridization
experiments had shown not to be concentrated in the
FIGURE 3. miRNA LNA in situ hybridization in L6 myoblasts. Both fluorescence (top) and phase (bottom) images of typical cells are shown for
each miRNA tested, with the name of the miRNA listed below the pair. Images are scaled to allow visualization of intranuclear localization
patterns and should not be used to compare relative quantities between microRNAs. Exposure times were 3 sec for miR-125, miR-199, let-7a,
miR-206, and miR-21; 500 msec for miR-351 and miR-664; 200 msec for miR-1 and miR-A; and 100 msec for miR-494. Bar, 3 mm.
Politz et al.
RNA, Vol. 15, No. 9
nucleolus (Fig. 3; Politz et al. 2006). These results corrob-
orated the microarray results, which indicated that these
miRNAs were not significantly nucleolus-concentrated,
although they have somewhat lower nucleolus/nucleoplasm
ratios in the in situ hybridization study than predicted by
the microarray data (data not shown). Importantly,
although these three miRNAs, miR-199a-3p, miR-125a-
5p, and let-7a, showed microarray nucleolus/nucleoplasm
ratios similar to miR-206 and miR-1, we did not observe
concentrated nucleolar signal in any cells hybridized with
probes to these miRNAs. This indicates that the group of
miRNAs showing similar levels of nucleolar and nucleo-
plasmic signal in the array represents miRNAs that show
nucleolar localization in some cells (e.g., miR-1 and miR-
206) as well as those that do not (e.g., miR-199a-3p, miR-
125a-5p, let-7a). Thus, it appears that microarray analysis
alone gives a low estimate of the number of nucleolus-
concentrated miRNAs in a population.
Figure 3 also shows that miR-A, a variant of rat miR-1
that contains a G-to-A substitution, did not display sub-
stantial nucleolar hybridization, verifying the specificity of
the LNA probe hybridization. All hybridization signals
shown in Figure 3, except for miR-21 (vide infra), were
RNase sensitive, indicating that the LNA probes are
targeting RNA (Supplemental Fig. 2; Politz et al. 2006).
As just mentioned, miR-21 unexpectedly showed no
significant RNase-sensitive in situ hybridization signal (Fig.
3; Supplemental Fig. 2) even though high levels of this
microRNA were detected in all cell compartments in the
types besides muscle and its deregulated expression has been
linked to neoplasia (Calin et al. 2005; Tong and Nemunaitis
a protein (or RNA or other molecule) in rat myoblasts that
blocks its in situ hybridization. This interpretation was
substantiated by RT-qPCR analyses below.
Since the above results demonstrated that a number of
miRNAs were concentrated in the nucleolus, we wished to
learn whether these miRNAs were present in precursor or
mature form and also determine their relative amounts
compared with one another. This information cannot be
gleaned from the microarray and in situ analyses because
the LNA probes were designed to detect all miRNA forms
and possessed potentially different affinities for their target
miRNAs. Therefore, we used TaqMan RT-qPCR to compare
enriched RNA fractions isolated from L6 myoblasts. TaqMan
RT-qPCR selectively amplifies mature miRNAs because the
primers do not hybridize appreciably or at all to miRNA
precursor forms (Chen et al. 2005; Schmittgen et al. 2008).
Figure 4 shows the relative amounts of the nucleolus-
concentrated miRNAs detected using this method. Levels of
the E2 snoRNA and Y1 Ro RNA controls provide an estima-
tion of the relative levels of a nucleolus-concentrated and a
nucleolus-depleted small RNA, respectively (see also Fig. 1C).
Both miR-206 and miR-21 were detected in the nucleolus-
enriched fraction at levels higher even than the E2 snoRNA,
which strongly suggests that high levels of the mature form
of these two miRNAs are present in the nucleolus. These
results substantiate our earlier findings for miR-206 (Politz
et al. 2006). As discussed above, miR-21 was not detected in
situ using the LNA probe, but it was detected at high levels
in the microarray assay (in all compartments) (Fig. 2B; data
not shown), which is consistent with these RT-qPCR
results. Taken together, a simple interpretation of these
results is that miR-21 is present as a mature miRNA in all
three compartments but is somehow masked from detec-
tion by in situ hybridization. miR-1 and miR-351 were
detected by RT-qPCR at lower levels than the nucleolus-
abundant snoRNA but, nevertheless, were present at levels
higher than Y1, the predominantly cytoplasmic RNA
species that represents the ‘‘background’’ in the nucleolar
preparation. The RT-qPCR thus confirms that these two
miRNAs are present in significant amounts in the nucle-
olus, although the levels of the mature forms of these
miRNAs are considerably lower than miR-206 and miR-21.
Finally, miR-494 and miR-664 were detected by RT-qPCR
of nucleolar RNA at levels similar to or below that of Y1
RNA, which indicates that the mature forms of these two
miRNAs are not concentrated in the nucleolus. Since both
these miRNAs were observed at significant levels in the
nucleolus in the microarray analysis and by in situ hybrid-
ization (where the LNA probes detect both mature and
FIGURE 4. Detection of mature miRNAs in the nucleolus. TaqMan
RT-qPCR microRNA assays were employed to characterize and
quantify miRNAs identified in the nucleolus-enriched fraction of L6
myoblasts. In this method, a hairpin primer selectively hybridizes to a
particular mature miRNA target and primes reverse transcription of
only that mature miRNA and not its precursor or primary transcript
within an RNA fraction. The RT product is then amplified using
miRNA-specific PCR primers, and the number of copies is measured
by hybridization of a specific fluorescent probe as real time PCR
proceeds (Chen et al. 2005; Schmittgen et al. 2008). Relative copy
numbers were normalized between two experiments using parallel RT
reactions that were spiked with a control RNA, IPC (see Materials and
Methods). The graph illustrates the levels of mature miRNAs relative
to one another (in arbitrary units expressed as DCtin log base 2) in
nucleolus-enriched fractions. The y intercept for the x axis has been
set at the value for Y1 (a prevalent cytoplasmic small RNA), which
represents background. Also shown are results for snoRNA E2, a small
RNA that is highly concentrated in the nucleolus. Error bars, SE
between the two experiments.
miRNAs in the nucleolus
precursor forms), taken together these results suggest that
these miRNAs are present in precursor form in the nucleolus.
As mentioned in the Introduction, the nucleolus has a
tripartite organization consisting of fibrillar centers, the DFC
and the GC (Huang 2002; Raska et al. 2006). From the
results in Figure 3 and as shown for miR-206 earlier (Politz
et al. 2006), we knew that the newly identified nucleolar
miRNAs did not localize exclusively to the small, focal
fibrillar centers. In order to determine the distribution of
these miRNAs with respect to the DFC and GC, we first
immunostained cells for fibrillarin, a fiduciary marker of the
DFC, and then performed in situ hybridization with LNA
probes for miR-351, miR-494, or miR-664. The distribution
of each miRNA within the nucleolus was compared to that
of fibrillarin after deconvolution of optical stacks (Fig. 5). All
three miRNAs showed highest signals at regions that did not
overlap with the DFC and thus were primarily concentrated
in the GC. The miR-351 signal was most restricted to the
GC, while miR-494 and especially miR-664 showed addi-
tional signal in the DFC (note intensity line scans in Fig. 5).
Therefore, as previously reported for miR-206 (Politz et al.
2006), the most concentrated signal for all three nucleolar-
concentrated miRNAs was localized in the GC, the com-
partment in which other nonribosomal components con-
gress (Ma and Pederson 2008b).
We show here that many miRNAs are present in the
nucleolus of rat myoblasts and that some of these miRNAs
are concentrated therein. Our data suggest that some
miRNAs are present in the nucleolus in precursor form
(miR-494 and miR-664), while others are present as mature
miRNAs (miR-206, miR-21, miR-1, and miR-351), and
that members of both groups concentrate in the same
subnucleolar compartment, the GC. This compartment
also harbors various nonribosomal nucleolar components
discovered recently (Karayan et al. 2001; Politz et al. 2002,
2005; Ma and Pederson 2008a).
It is still early days in the microRNA field, and there are
many possible roles that nucleolar association might play in
the life cycle of a miRNA. Our results allow a clearer de-
lineation of the possibilities. First, none of the five miRNAs
that showed the highest nucleolar concentration using the
microarray assay, miR-340-5p, miR-351, miR-494, miR-
664, or let-7e, are thought to be skeletal muscle-specific
miRNAs (i.e., miR-1, miR-133, and miR-206 and perhaps
miR-95, miR-128a, and miR-499) (Lee et al. 2008; McCarthy
2008). It is thus clear that non-muscle-specific miRNAs
concentrate in the nucleolus of L6 myoblasts. Furthermore,
we found that the majority of the miRNAs expressed in
myoblasts (which includes ubiquitously expressed miRNAs)
can be detected in all three subcellular compartments (see
Supplemental Table 1), so our data do not support a model
in which myoblast-enriched miRNAs alone exhibit a
nucleolar presence. It also does not appear that there is a
class of ‘‘nucleolar’’ miRNAs that persists across tissue
types because in the myoblast cells we used, we do not see a
significant nucleolar presence of some miRNAs shown (by
others concurrently with this study) to be nucleolar in
testes, e.g., miR-127 (Robertus et al. 2009) and miR-214
(Marcon et al. 2008).
Other small RNAs are present in the nucleolus for
processing/modification or assembly into ribonucleopro-
tein complexes or are active in the nucleolus as regulatory
modification/processing components themselves (Politz
et al. 2002; Kiss et al. 2006; Raska et al. 2006; Boisvert
et al. 2007; Matera et al. 2007). A nucleolar role in pri-
miRNA processing or modification is possible, although it
has been shown recently that the processing of at least
some pri-miRNAs is linked to their transcription and thus
occurs at sites of pol II activity and not the nucleolus
(Morlando et al. 2008; Pawlicki and Steitz 2008). However,
it has also been reported that Drosha associates with the
nucleolus (Shiohama et al. 2007), and moreover, a non-
coding RNA that is processed by Drosha has been shown to
associate with the nucleolus (Ganesan and Rao 2008).
Furthermore, some miRNAs are processed from mirtrons
in a Drosha-independent manner (Berezikov et al. 2007),
and most intriguingly, there is very recent evidence that
snoRNAs may be precursors to some miRNAs and/or small
FIGURE 5. Subnucleolar mapping of miRNAs. L6 myoblasts were
grown to z70% confluence; fixed and subjected to immunostaining
for fibrillarin, a fiduciary marker for the nucleolar DFC; followed by
detection of miRNAs by in situ hybridization. Optical stacks were
then captured, deconvolved, and color-merged. Images show magni-
fied nucleoli cropped from a central plane of deconvolved stacks. (A)
miR-351; (E) miR-494; (I) miR-664. (B,F,J) Fibrillarin. (C,G,K) Color
merge of two images in row. The white line across the nucleolus
indicates the position of the densitometric line scans shown in D, H,
and L, respectively. Line scans indicate the relative intensities of
fibrillarin (green) and the miRNA of interest (red). Scale bars, 0.5 mm.
Politz et al.
RNA, Vol. 15, No. 9
RNAs (Ender et al. 2008; Saraiya and Wang 2008; Taft et al.
2009). Indeed, it has been reported that the small nucleolar
RNA, SNORA36B/ACA36b, which was identified as a
GAR1-binding snoRNA that is predicted to guide pseudo-
uridylation of 18S rRNA in the nucleolus (Kiss et al.
2004), is a precursor to miR-664 (Ender et al. 2008).
Nothing is known about the mechanism of miRNA
processing from a snoRNA, although it is certainly possible
that processing occurs in the nucleolus, since snoRNAs
accumulate there. Our results suggest that miR-664 is pre-
sent in the nucleolus as a precursor, which is consistent
with this model. Since we did not see the most concen-
trated miR-664 signal in the DFC, where it might be ex-
pected if the LNA probe were hybridizing to a SNORA36B/
ACA36b-GAR1 complex, more work will be necessary to
determine the compartment and mechanism of this puta-
tive processing step. However, no matter how inviting the
hypothesis may be that all nucleolus-localized miRNAs
have snoRNA precursors, we have found no evidence for
complete identity between the sequences of the other
miRNAs we have characterized here and any rat snoRNA
or rRNA sequences. (Homology searches were performed
using the Toulouse human snoRNA database [http://www-
snorna.biotoul.fr/index.php] in conjunction with Ensembl
[http://www.ensembl.org/ and ncRNA BLAT at http://www.
ensembl.org/Multi/blastview] and USCS [http://genome.
ucsc.edu/] genome browsers to test human/rat alignments).
It should be pointed out here that cross-hybridization of
the miR-664 LNA probe to SNORA36B would occur
regardless of the precursor nature of this molecule, but
we observe, at most, only partial homology of other
miRNAs to snoRNAs, which can occur randomly and
would not support cross-hybridization of the highly spe-
cific LNA probes. Thus, our data do not currently support a
model that all nucleolar-localized miRNAs are processed
from snoRNA precursors.
It is also possible that some miRNAs traffic to the
nucleolus for modification. Significantly, we note that
A/I editing of non-miRNAs by the adenosine deaminase,
ADAR2, has been shown to occur in the nucleolus (Vitali
et al. 2005), and it is known that some miRNA precursors
are subject to editing (Luciano et al. 2004; Blow et al. 2006;
Habig et al. 2007; Kawahara et al. 2008). miRNA editing
can also interfere with processing (Yang et al. 2006;
Kawahara et al. 2007), which conceivably could cause
sequestration of unprocessed miRNA precursors in the
nucleolus. The spatiotemporal aspects of small RNA editing
requires more study, however, since another adenosine
deaminase, ADAR1, has been shown to bind (but not
modify) dsRNA and inhibit RNAi in the cytoplasm in
mammals (Yang et al. 2005).
Rather than RNA processing or modification, it may be
that certain miRNAs visit the nucleolus to assemble
with the appropriate proteins and/or RNAs necessary for
transport of the active miRNP from the nucleus. The
signal recognition particle RNA traffics through the nucle-
olus to assemble with specific proteins (Jacobson and
Pederson 1998; Politz et al. 2000; Sommerville et al.
2005) and, in the process, accumulates in the GC
(Politz et al. 2002), just as do the nucleolus-concentrated
miRNAs. We also speculate that some miRNAs could
combine with target messenger RNAs in the nucleolus to
be exported as ‘‘presuppressed’’ mRNAs, a somatic cell
analogy with masked maternal mRNAs in oocytes. Consis-
tent with this idea, there is evidence that the nucleolus is
involved in mRNA nucleocytoplasmic transport (Schneiter
et al. 1995; Thomsen et al. 2008), and some mRNAs have
been shown to associate with the nucleolus (John et al.
1977; Bond and Wold 1993; Ba ´rtova ´ et al. 2008; Kim et al.
Finally, miRNAs may concentrate in the nucleolus be-
cause they are active as regulatory modification/processing
components themselves. For example, small noncoding
RNAs have been implicated in the control of ribosomal
DNA transcription (Mayer et al. 2008), and snoRNAs guide
modification of rRNA and some snRNAs in the nucleolus
(Boisvert et al. 2007; Matera et al. 2007), as well as perhaps
modulating the activity of ADAR2 (Vitali et al. 2005).
However, neither miR-206 nor the nucleolus-enriched
miRNAs identified in the present study were localized at
the rDNA transcription sites (fibrillar centers) or to the
DFC, where most snoRNA-guided rRNA modifications are
thought to occur, but rather were clustered in the GC, a
region that is known to be the site of late stages of rRNA
processing. Furthermore, homology searches revealed that,
except for miR-664, the sequences of these miRNAs do not
share identity with any annotated snoRNA-like sequences
necessary to guide 18S or 28S rRNA modifications, al-
though we did find that miRNA-199a-3p and miR-494 pos-
sess 9–10 nucleotide (nt) sequences that are complementary
to regions immediately adjacent to the guide sequences of
SNORA61 and SNORA60, respectively (data not shown). If
miRNAs hybridized to these regions, this could interfere
with snoRNA-guided modification of target RNAs. How-
ever, our results suggest that miR-494 is present as a pre-
cursor in the nucleolus and we did not observe similar
complementarity for any other miRNAs with any other
snoRNAs, although we point out that the rat snoRNAs are
not as well annotated as the human/yeast snoRNAs so this
search is presently incomplete. If miRNAs do perform this
and/or other functions in the nucleolus, they presumably
would do this in addition to their function in gene silencing
at the translational level in the cytoplasm.
In summary, we find it unlikely that only myoblast-
specific miRNAs accumulate in nucleoli and likely that at
least some miRNAs arise from snoRNA precursors. It
remains possible that some miRNAs visit the nucleolus
for modification, assembly with RNA/protein partners, or
regulation of snoRNA activity. In these conceptions of the
possible functional significance of the presence—and in
miRNAs in the nucleolus
some cases a concentration of—these miRNAs in the
nucleolus, we again note that mature miRNAs and other
small RNAs can return to the nucleus from the cytoplasm
(Hwang et al. 2007; Guang et al. 2008; Kim et al. 2008;
Marcon et al. 2008; Ohrt et al. 2008; Place et al. 2008). The
fact that even the first translational RNA discovered,
transfer RNA, also is known to display this beguiling
‘‘retrograde’’ transit in both yeast (Shaheen and Hopper
2005; Takano et al. 2005) and mammalian cells (Shaheen
et al. 2007) invites the consideration of broader, evolution-
ary possibilities for the nuclear and nucleolar presence of
microRNAs. We have demonstrated that a large number of
miRNAs are present in the nucleolus, rather than a select
few, so any models to explain a role for nucleolar miRNAs
must apply to a general miRNA population. We have also
carried out preliminary miRNA LNA in situ hybridization
experiments in HeLa cells and have found that some of the
miRNAs that concentrated in the nucleoli of rat myoblasts
also concentrate in the nucleolus of these cancer cells (not
shown), giving further weight to the notion that this
localization is a general phenomenon. Further work is
now necessary to define the role that nucleolar transit plays
in the life cycle of miRNAs.
MATERIALS AND METHODS
L6 myoblasts were grown in DMEM with 10% fetal bovine serum
at 37°C with 5% CO2to 80% subconfluence in ten 225-cm2flasks
(yielding z108cells), harvested using trypsin, and then washed
three times with DMEM salts. Cells were resuspended in a
hypotonic buffer (Mellon and Bhorjee 1982), allowed to swell,
and then broken using a Dounce homogenizer with a clearance of
z50 mm (Penman et al. 1966). Nuclei were purified through a
sucrose cushion (Andersen et al. 2002) and sonicated (Pederson
1974), and the nucleoli were pelleted (Andersen et al. 2002).
During isolation and washing, purity of nucleoli was judged by
light microscopy. Total RNA was isolated from the nucleo-
lar, nucleoplasmic, and cytoplasmic fractions using a Qiagen
miRNAeasy kit. The resulting RNA is enriched in all the cell’s
small RNA species, including miRNAs <200 nt.
Two-color microarray analysis was performed using an LNA-
based miCHIP (miRCURY microarrays, Exiqon) to screen for the
full complement of rat miRNAs recorded in miRBase (version 9.2,
Wellcome Trust Sanger Institute). Five to eight micrograms of
total RNA from each subcellular fraction described above was
labeled with Hy3 (a proprietary Cy3-like dye of Exiqon), and
additionally, equal aliquots of all three fractions were pooled and
labeled with Hy5 (Exiqon). This pooled ‘‘total RNA’’ was used for
normalization in all runs. Four replicate two-color runs were
performed for each fraction, and the results were background
subtracted and subjected to a Loess and quantile normalization by
Exiqon’s in house profiling service. miRNAs showing significant
difference in nucleolar level were determined as those having a SD
$ 0.5 (see Supplemental Table 1).
In situ hybridization and immunostaining
In situ hybridization using specific miRNA LNA probes, image
capture, and analysis was as described (Politz et al. 2006). RNase
treatment was also as described (Politz et al. 2006). Immunostain-
ing followed by in situ hybridization was performed with a
fibrillarin monoclonal antibody as detailed previously (Politz
et al. 2002), except optical stacks were captured using a Deltavi-
sion workstation and deconvolved using Softworx. Control cells
subjected to either immunostaining or in situ hybridization alone
showed no bleed-through of signal into the other channel. Images
were adjusted for size and contrast and color merged using Image
J. The Image J plug-in, color profiler, was used to obtain line
The two-step TaqMan miRNA RT-qPCR system (Applied Bio-
systems) was used to follow miRNA amplification in real time
(Chen et al. 2005; Schmittgen et al. 2008). Reverse transcription
and PCR reactions were performed according to the manufac-
turer’s protocol using TaqMan rat stem-loop RT and PCR primers
and probes specific for each miRNA under study. A synthetic
exogenous internal positive control (IPC, Applied Biosystems no.
4308323) was spiked into parallel samples of total nucleolar RNA,
amplified using IPC-specific primers and TaqMan probes, and
used for normalization between PCR reactions. snoRNA E2
(U64702) (Selvamurugan et al. 1997) and the Ro family Y1
RNA controls were purchased from Applied Biosystems. Fluores-
cence was quantitated using an Applied Biosystems 7500 Fast
Real-Time PCR System.
Supplemental material can be found at http://www.rnajournal.org.
This work was supported by grant MCB-0445841 from the
National Science Foundation. We thank Michael Hansen at
Exiqon technical services (Vedbaek, Denmark) for help with
microarray data analysis and Mark Groudine and the Scientific
Imaging Group at the Fred Hutchinson Cancer Research Center
for the use of the Deltavision workstation. We also thank Paul
Gardner (University of Massachusetts Medical School) for the use
of his RT-qPCR thermal cycler, Hanhui Ma for help with cell
culture, and Denise Maclachlan for assistance in manuscript
Received November 17, 2008; accepted June 18, 2009.
Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H,
Mann M, Lamond AI. 2002. Directed proteomic analysis of the
human nucleolus. Curr Biol 12: 1–11.
Politz et al.
RNA, Vol. 15, No. 9
Ason B, Darnell DK, Wittbrodt B, Berezikov E, Kloosterman WP,
Wittbrodt J, Antin PB, Plasterk RH. 2006. Differences in verte-
brate microRNA expression. Proc Natl Acad Sci 103: 14385–
Ba ´rtova ´ E, Harnicarova ´ A, Krejcı ´ J, Strasa ´k L, Kozubek S. 2008. Single-
cell c-myc gene expression in relationship to nuclear domains.
Chromosome Res 16: 325–343.
Berezikov E, Chung WJ, Willis J, Cuppen E, Lai EC. 2007. Mammalian
mirtron genes. Mol Cell 28: 328–336.
Bhorjee JS, Pederson T. 1972. Nonhistone chromosomal proteins in
synchronized HeLa cells. Proc Natl Acad Sci 69: 3345–3349.
Bhorjee JS, Pederson T. 1973. Chromatin: Its isolation from cultured
mammalian cells with particular reference to contamination
by nuclear ribonucleoprotein particles. Biochemistry 12: 2766–
Blow, M.J., Grocock, R.J., van Dongen, S., Enright, A.J., Dicks, E.,
Futreal, P.A., Wooster, R., Stratton, M.R. 2006. RNA editing of
human microRNAs. Genome Biol 7: R27.1–R27.8.
Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. 2007.
The multifunctional nucleolus. Nat Rev Mol Cell Biol 8: 574–
Bond VC, Wold B. 1993. Nucleolar localization of myc transcripts.
Mol Cell Biol 13: 3221–3230.
Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE,
Iorio MV, Visone R, Sever NI, Fabbri M, et al. 2005. A microRNA
signature associated with prognosis and progression in chronic
lymphocytic leukemia. N Engl J Med 353: 1793–1801.
Carthew RW, Sontheimer EJ. 2009. Origins and mechanisms of
miRNAs and siRNAs. Cell 136: 642–655.
Castoldi M, Schmidt S, Benes V, Hentze MW, Muckenthaler MU.
2008. miChip: An array-based method for microRNA expression
profiling using locked nucleic acid capture probes. Nat Protocols 3:
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT,
Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, et al. 2005.
Real-time quantification of microRNAs by stem-loop RT-PCR.
Nucleic Acids Res 33: e179. doi: 10.1093/nar/gni178.
Du T, Zamore PD. 2005. microPrimer: The biogenesis and function of
microRNA. Development 132: 4645–4652.
Eisenberg I, Eran A, Nishino I, Moggio M, Lamperti C, Amato AA,
Lidov HG, Kang PB, North KN, Mitrani-Rosenbaum S, et al. 2007.
Distinctive patterns of microRNA expression in primary muscular
disorders. Proc Natl Acad Sci 104: 17016–17021.
Ender C, Krek A, Friedlander MR, Beitzinger M, Weinmann L,
Chen W, Pfeffer S, Rajewsky N, Meister G. 2008. A human
snoRNA with microRNA-like functions. Mol Cell 32: 519–528.
Filipowicz W, Bhattacharyya SN, Sonenberg N. 2008. Mechanisms of
post-transcriptional regulation by microRNAs: Are the answers in
sight? Nat Rev Genet 9: 102–114.
Ganesan G, Rao SM. 2008. A novel noncoding RNA processed
by Drosha is restricted to nucleus in mouse. RNA 14: 1399–
Ganot P, Jady BE, Bortolin ML, Darzacq X, Kiss T. 1999. Nucleolar
factors direct the 29-O-ribose methylation and pseudouridylation
of U6 spliceosomal RNA. Mol Cell Biol 19: 6906–6917.
Gerbi SA, Borovjagin AV, Lange TS. 2003. The nucleolus: A site of
ribonucleoprotein maturation. Curr Opin Cell Biol 15: 318–325.
Guang S, Bochner AF, Pavelec DM, Burkhart KB, Harding S,
Lachowiec J, Kennedy S. 2008. An Argonaute transports siRNAs
from the cytoplasm to the nucleus. Science 321: 537–541.
Habig JW, Dale T, Bass BL. 2007. miRNA editing—we should have
inosine this coming. Mol Cell 25: 792–793.
Hendrick JP, Wolin SL, Rinke J, Lerner MR, Steitz JA. 1981. Ro small
cytoplasmic ribonucleoproteins are a subclass of La ribonucleo-
proteins: Further characterization of the Ro and La small ribonu-
cleoproteins from uninfected mammalian cells. Mol Cell Biol 1:
Huang S. 2002. Building an efficient factory: Where is pre-rRNA
synthesized in the nucleolus? J Cell Biol 157: 739–741.
Hwang HW, Wentzel EA, Mendell JT. 2007. A hexanucleotide element
directs microRNA nuclear import. Science 315: 97–100.
Jacobson MR, Pederson T. 1998. Localization of signal recognition
particle RNA in the nucleolus of mammalian cells. Proc Natl Acad
Sci 95: 7981–7986.
John HA, Patrinou-Georgoulas M, Jones KW. 1977. Detection of
myosin heavy chain mRNA during myogenesis in tissue culture by
in vitro and in situ hybridization. Cell 12: 501–508.
Karayan L, Riou JF, Seite P, Migeon J, Cantereau A, Larsen CJ. 2001.
Human ARF protein interacts with topoisomerase I and stimulates
its activity. Oncogene 20: 836–848.
Kawahara Y, Zinshteyn B, Chendrimada TP, Shiekhattar R,
Nishikura K. 2007. RNA editing of the microRNA-151 precursor
blocks cleavage by the Dicer–TRBP complex. EMBO Rep 8: 763–769.
Hatzigeorgiou AG, Nishikura K. 2008. Frequency and fate of
microRNA editing in human brain. Nucleic Acids Res 36: 5270–
Kim J, Krichevsky A, Grad Y, Hayes GD, Kosik KS, Church GM,
Ruvkun G. 2004. Identification of many microRNAs that copurify
with polyribosomes in mammalian neurons. Proc Natl Acad Sci
Kim HK, Lee YS, Sivaprasad U, Malhotra A, Dutta A. 2006. Muscle-
specific microRNA miR-206 promotes muscle differentiation. J
Cell Biol 174: 677–687.
Kim DH, Saetrom P, Snove O Jr, Rossi JJ. 2008. MicroRNA-directed
transcriptional gene silencing in mammalian cells. Proc Natl Acad
Sci 105: 16230–16235.
Kim SH, Koroleva OA, Lewandowska D, Pendle AF, Clark GP,
Simpson CG, Shaw PJ, Brown JWS. 2009. Aberrant mRNA
transcripts and the nonsense-mediated decay proteins UPF2 and
UPF3 are enriched in the Arabidopsis nucleolus. Plant Cell 21: (in
press). doi: 10.1105/tpc.109.067736.
Kiss AM, Jady BE, Bertrand E, Kiss T. 2004. Human box H/ACA
pseudouridylation guide RNA machinery. Mol Cell Biol 24: 5797–
Kiss T, Fayet E, Jady BE, Richard P, Weber M. 2006. Biogenesis and
intranuclear trafficking of human box C/D and H/ACA RNPs.
Cold Spring Harb Symp Quant Biol 71: 407–417.
Lee EJ, Baek M, Gusev Y, Brackett DJ, Nuovo GJ, Schmittgen TD.
2008. Systematic evaluation of microRNA processing patterns in
tissues, cell lines, and tumors. RNA 14: 35–42.
Luciano DJ, Mirsky H, Vendetti NJ, Maas S. 2004. RNA editing of a
miRNA precursor. RNA 10: 1174–1177.
Ma H, Pederson T. 2008a. Nucleophosmin is a binding partner of
nucleostemin in human osteosarcoma cells. Mol Biol Cell 19:
Ma H, Pederson T. 2008b. Nucleostemin: A multiplex regulator of
cell-cycle progression. Trends Cell Biol 18: 575–579.
Maggio R, Siekevitz P, Palade GE. 1963. Studies in isolated nuclei. I.
Isolation and chemical characterization of a nuclear fraction from
guinea pig liver. J Cell Biol 18: 267–291.
Marcon E, Babak T, Chua G, Hughes T, Moens PB. 2008. miRNA and
piRNA localization in the male mammalian meiotic nucleus.
Chromosome Res 16: 243–260.
Matera AG, Tycowski KT, Steitz JA, Ward DC. 1994. Organization of
small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in
situ hybridization and immunocytochemistry. Mol Biol Cell 5:
Matera AG, Terns RM, Terns MP. 2007. Non-coding RNAs: Lessons
from the small nuclear and small nucleolar RNAs. Nat Rev Mol
Cell Biol 8: 209–220.
Matranga C, Zamore P. 2007. Small silencing RNAs. Curr Biol 17:
Mayer C, Neubert M, Grummt I. 2008. The structure of NoRC-
associated RNA is crucial for targeting the chromatin remodelling
complex NoRC to the nucleolus. EMBO Rep 9: 774–780.
McCarthy JJ. 2008. MicroRNA-206: The skeletal muscle-specific
myomiR. Biochim Biophys Acta 1779: 682–691.
miRNAs in the nucleolus
Mellon I, Bhorjee J. 1982. Isolation and characterization of nuclei and
purification of chromatin from differentiating cultures of rat
skeletal muscle. Exp Cell Res 137: 141–154.
Mishra RK, Eliceiri GL. 1997. Three small nucleolar RNAs that are
involved in ribosomal RNA precursor processing. Proc Natl Acad
Sci 94: 4972–4977.
Morlando M, Ballarino M, Gromak N, Pagano F, Bozzoni I,
Proudfoot NJ. 2008. Primary microRNA transcripts are processed
co-transcriptionally. Nat Struct Mol Biol 15: 902–909.
Muramatsu M, Smetana K, Busch H. 1963. Quantitative aspects of
isolation of nucleoli of Walker carcinosarcoma and liver of the rat.
Cancer Res 23: 510–518.
Newman MA, Thomson JM, Hammond SM. 2008. Lin-28 interaction
with the Let-7 precursor loop mediates regulated microRNA
processing. RNA 14: 1539–1549.
Ochs RL, Lischwe MA, Spohn WH, Busch H. 1985. Fibrillarin: A new
protein of the nucleolus identified by autoimmune sera. Biol Cell
Ohrt T, Mutze J, Staroske W, Weinmann L, Hock J, Crell K,
Meister G, Schwille P. 2008. Fluorescence correlation spectroscopy
and fluorescence cross-correlation spectroscopy reveal the cyto-
plasmic origination of loaded nuclear RISC in vivo in human cells.
Nucleic Acids Res 36: 6439–6449.
Pawlicki JM, Steitz JA. 2008. Primary microRNA transcript retention
at sites of transcription leads to enhanced microRNA production. J
Cell Biol 182: 61–76.
Pederson T. 1974. Proteins associated with heterogeneous nuclear
RNA in eukaryotic cells. J Mol Biol 83: 163–183.
Pederson T. 1998. Growth factors in the nucleolus? J Cell Biol 143:
Pederson T, Tsai RY. 2009. In search of nonribosomal nucleolar
protein function and regulation. J Cell Biol 184: 771–776.
Penman S, Smith I, Holtzman E. 1966. Ribosomal RNA synthesis and
processing in a particulate site in the HeLa cell nucleus. Science
Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R. 2008. MicroRNA-
373 induces expression of genes with complementary promoter
sequences. Proc Natl Acad Sci 105: 1608–1613.
Pogacic V, Dragon F, Filipowicz W. 2000. Human H/ACA small
nucleolar RNPs and telomerase share evolutionarily conserved
proteins NHP2 and NOP10. Mol Cell Biol 20: 9028–9040.
Politz J, Yarovoi S, Kilroy S, Gowda K, Zwieb C, Pederson T. 2000.
Signal recognition particle components in the nucleolus. Proc Natl
Acad Sci 97: 55–60.
Politz J, Lewandowski L, Pederson T. 2002. Signal recognition particle
RNA localization within the nucleolus differs from the classical
sites of ribosome synthesis. J Cell Biol 159: 411–418.
Politz J, Polena I, Trask I, Bazett-Jones D, Pederson T. 2005. A
nonribosomal landscape in the nucleolus revealed by the stem cell
protein nucleostemin. Mol Biol Cell 16: 3401–3410.
Politz JC, Zhang F, Pederson T. 2006. MicroRNA-206 colocalizes with
ribosome-rich regions in both the nucleolus and cytoplasm of rat
myogenic cells. Proc Natl Acad Sci 103: 18957–18962.
Raska I, Shaw PJ, Cmarko D. 2006. New insights into nucleolar
architecture and activity. Int Rev Cytol 255: 177–235.
Robertus JL, Harms G, Blokzijl T, Booman M, de Jong D, van
Imhoff G, Rosati S, Schuuring E, Kluin P, van den Berg A. 2009.
Specific expression of miR-17-5p and miR-127 in testicular and
central nervous system diffuse large B-cell lymphoma. Mod Pathol
Rosenberg MI, Georges SA, Asawachaicharn A, Analau E, Tapscott SJ.
2006. MyoD inhibits Fstl1 and Utrn expression by inducing
transcription of miR-206. J Cell Biol 175: 77–85.
Saraiya AA, Wang CC. 2008. snoRNA, a novel precursor of microRNA
in Giardia lamblia. PLoS Pathog 4: e1000224. doi: 10.1371/
Schmittgen TD, Lee EJ, Jiang J, Sarkar A, Yang L, Elton TS, Chen C.
2008. Real-time PCR quantification of precursor and mature
microRNA. Methods 44: 31–38.
Schneiter R, Kadowski T, Tartakoff AM. 1995. mRNA transport in
yeast: Time to reinvestigate the functions of the nucleolus. Mol
Biol Cell 6: 357–370.
Selvamurugan N, Joost OH, Haas ES, Brown JW, Galvin NJ,
Eliceiri GL. 1997. Intracellular localization and unique conserved
sequences of three small nucleolar RNAs. Nucleic Acids Res 25:
Dmitrovsky, E., and Ambros, V. 2004. Expression profiling of
mammalian microRNAs uncovers a subset of brain-expressed
microRNAs with possible roles in murine and human neuronal
differentiation. Genome Biol. 5: R13.1–R13.11.
Shaheen HH, Hopper AK. 2005. Retrograde movement of tRNAs
from the cytoplasm to the nucleus in Saccharomyces cerevisiae. Proc
Natl Acad Sci 102: 11290–11295.
Shaheen HH, Horetsky RL, Kimball SR, Murthi A, Jefferson LS,
Hopper AK. 2007. Retrograde nuclear accumulation of cytoplas-
mic tRNA in rat hepatoma cells in response to amino acid
deprivation. Proc Natl Acad Sci 104: 8845–8850.
Shiohama A, Sasaki T, Noda S, Minoshima S, Shimizu N. 2007.
Nucleolar localization of DGCR8 and identification of eleven
DGCR8-associated proteins. Exp Cell Res 313: 4196–4207.
Sommerville J, Brumwell C, Politz J, Pederson T. 2005. Signal
recognition particle assembly in relation to the function
of amplified nucleoli of Xenopus oocytes. J Cell Sci 118: 1299–
Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T,
Dalmay T, Munsterberg A. 2008. Specific requirements of MRFs
for the expression of muscle specific microRNAs, miR-1, miR-206,
and miR-133. Dev Biol 321: 491–499.
Taft RJ, Glazov EA, Lassmann T, Hayashizaki Y, Carninci P,
Mattick JS. 2009. Small RNAs derived from snoRNAs. RNA 15:
Takano A, Endo T, Yoshihisa T. 2005. tRNA actively shuttles between
the nucleus and cytosol in yeast. Science 309: 140–142.
Thomsen R, Nielsen PS, Jensen TH. 2005. Dramatically improved
RNA in situ hybridization signals using LNA-modified probes.
RNA 11: 1745–1748.
Thomsen R, Saguez C, Nasser T, Jensen TH. 2008. General, rapid, and
transcription-dependent fragmentation of nucleolar antigens in S.
cerevisiae mRNA export mutants. RNA 14: 706–716.
Thomson JM, Newman M, Parker JS, Morin-Kensicki EM, Wright T,
Hammond SM. 2006. Extensive post-transcriptional regulation of
microRNAs and its implications for cancer. Genes & Dev 20: 2202–
Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M, Galuppo P,
Just S, Rottbauer W, Frantz S, et al. 2008. MicroRNA-21 contrib-
utes to myocardial disease by stimulating MAP kinase signalling in
fibroblasts. Nature 456: 980–984.
Tong AW, Nemunaitis J. 2008. Modulation of miRNA activity in
human cancer: A new paradigm for cancer gene therapy? Cancer
Gene Ther 15: 341–355.
van Rooij E, Sutherland LB, Liu N, Williams AH, McAnally J,
Gerard RD, Richardson JA, Olson EN. 2006. A signature pattern
of stress-responsive microRNAs that can evoke cardiac hypertro-
phy and heart failure. Proc Natl Acad Sci 103: 18255–18260.
van Rooij E, Liu N, Olson EN. 2008. MicroRNAs flex their muscles.
Trends Genet 24: 159–166.
Visintin R, Amon A. 2000. The nucleolus: The magician’s hat for cell
cycle tricks. Curr Opin Cell Biol 12: 752.
Vitali P, Basyuk E, Le Meur E, Bertrand E, Muscatelli F, Cavaille J,
Huttenhofer A. 2005. ADAR2-mediated editing of RNA substrates
in the nucleolus is inhibited by C/D small nucleolar RNAs. J Cell
Biol 169: 745–753.
Wang B, Yanez A, Novina CD. 2008. MicroRNA-repressed mRNAs
contain 40S but not 60S components. Proc Natl Acad Sci 105:
Wulczyn FG, Smirnova L, Rybak A, Brandt C, Kwidzinski E,
Ninnemann O, Strehle M, Seiler A, Schumacher S, Nitsch R.
Politz et al.
RNA, Vol. 15, No. 9
2007. Post-transcriptional regulation of the let-7 microRNA Download full-text
during neural cell specification. FASEB J 21: 415–426.
Yang W, Wang Q, Howell KL, Lee JT, Cho DS, Murray JM,
Nishikura K. 2005. ADAR1 RNA deaminase limits short interfer-
ing RNA efficacy in mammalian cells. J Biol Chem 280: 3946–
Yang W, Chendrimada TP, Wang Q, Higuchi M, Seeburg PH,
Shiekhattar R, Nishikura K. 2006. Modulation of microRNA
processing and expression through RNA editing by ADAR
deaminases. Nat Struct Mol Biol 13: 13–21.
Yeom KH, Lee Y, Han J, Suh MR, Kim VN. 2006. Characterization of
DGCR8/Pasha, the essential cofactor for Drosha in primary
miRNA processing. Nucleic Acids Res 34: 4622–4629.
Zeng Y, Yi R, Cullen BR. 2005. Recognition and cleavage of primary
microRNA precursors by the nuclear processing enzyme Drosha.
EMBO J 24: 138–148.
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