SNPs in human miRNA genes affect biogenesis
GUIHUA SUN,1,2JIN YAN,3KATIE NOLTNER,3JINONG FENG,3HAITANG LI,1DANIEL A. SARKIS,4
STEVE S. SOMMER,3,5and JOHN J. ROSSI1
1Department of Molecular Biology, City of Hope National Medical Center, Duarte, California 91010, USA
2Graduate School of Biological Science, City of Hope National Medical Center, Duarte, California 91010, USA
3Department of Molecular Genetics, City of Hope National Medical Center, Duarte, California 91010, USA
4Ruth and Eugene Roberts Summer Student Academy, City of Hope National Medical Center, Duarte, California 91010, USA
MicroRNAs (miRNAs) are 21–25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs
derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in
the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence
variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have
profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and
Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these
variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs
and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of
processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature
miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs
examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.
Keywords: microRNA; miRNA; SNP; miRNA-polymorphism; miR-SNP; miR-TS-SNP
MicroRNAs (miRNAs) are emerging as one of the most
interesting small regulatory, noncoding RNAs in molecular
biology. They are single stranded, about 22-nucleotides (nt)
alent to those of transcription factors by switching off
or fine tuning target gene expression. It is believed that they
function largely, though not exclusively, through base pair-
ing to the complementary sequences in the 39 untranslated
region (39UTR) of target genes to suppress translation.
Generally, they can repress translation by guiding the
miRNA-mediated RNA-induced silencing complex (miRISC)
binding to the 39UTR of a complementary message, disrupt-
ing translation initiation or elongation (Filipowicz et al.
2008). Several lines of evidence suggest that they may
perform their function in the RNA processing bodies
(P-bodies) by sequestering target transcripts to P-bodies for
storage, decapping, deadenylation, and degradation (Liu et al.
2005; Rehwinkel et al. 2005; Chu and Rana 2006).
miRNA genes are scattered among each of the chromo-
somes in humans, except for the Y chromosome. They
primarily derive from intronic or exonic capped, polyade-
nylated RNA polymerase II transcripts, termed ‘‘primary’’
miRNAs (pri-miRNA) (Lee et al. 2002, 2003; Cai et al.
2004). Mature miRNAs are generated by a two-step
processing mechanism (Supplemental Fig. S1). pri-miRNAs
are first processed to ‘‘hairpin-like,’’ partially duplexed
‘‘precursor’’ miRNAs (pre-miRNA) in the nucleus. Aside
from a small group of pre-miRNAs that are generated
through mRNA splicing/debranching machinery termed
the ‘‘miRtron pathway’’ (Berezikov et al. 2007; Okamura
et al. 2007; Ruby et al. 2007), most pre-miRNAs are
processed from pri-miRNAs by the nuclear ribonuclease
(RNase) III Drosha, which partners with the RNA-binding
protein DiGeorge syndrome critical region gene 8 (DGCR8)
(Lee et al. 2003; Han et al. 2004). pre-miRNAs are typically
5Present address: Medomics, Inc., Azusa, California 91702, USA.
Reprint requests to: John J. Rossi, Department of Molecular Biology,
City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA
91010-3000, USA; e-mail: email@example.com; fax: (626) 301-8271.
Article published online ahead of print. Article and publication date are
RNA (2009), 15:1640–1651. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2009 RNA Society.
55–80 nt in length and are exported to the cytoplasm by
exportin-5/RAN-GTP (Yi et al. 2003). The pre-miRNAs are
processed into z21–22-nt long miRNA/miRNA* duplexes
by RNase III Dicer, which partners with the RNA-binding
protein transactivation response (TAR) element RNA-
binding protein (TRBP) (Chendrimada et al. 2005; Haase
et al. 2005). The production of miRNA/miRNA* duplexes
is an essential step in miRNA biogenesis and precisely
defines the ends of the mature miRNAs for preferential
loading of the guide strand (Khvorova et al. 2003). The
choice of the guide strand is dependent in part on the
thermodynamic end properties of the duplex, with the least
thermodynamically stable 59 end usually being chosen as
the guide strand, while the other strand, labeled miRNA*, is
usually degraded (Ruvkun 2001; Hutvagner and Zamore
2002; Khvorova et al. 2003; Schwarz et al. 2003). Most
recently, the fates of the miRNA guide and miRNA* strands
have been shown to be tissue dependent (Ro et al. 2007),
with both strands being functionally active under specific
conditions (Okamura et al. 2008). Argonaute-mediated
loading into the processing complex can increase the bias
of strand loading (Seitz et al. 2008), and RNA-binding
proteins can selectively block the processing of pri-miRNAs
(Piskounova et al. 2008; Viswanathan et al. 2008).
The mature miRNAs are used to guide miRISC to the
complementary sequences in the 39UTR of targeted tran-
scripts. The result is site-specific mRNA cleavage when
the pairing is nearly complete (mostly in plants, rare in
animals) or translational inhibition when imperfect base
pairing occurs (mostly in animals) (Bartel 2004). For trans-
lational suppression, Watson–Crick base pairing between
six or seven consecutive nucleotides in the target mRNA’s
39UTR and nucleotides 2–7 or 2–8 (the ‘‘seed sequence’’) of
the miRNA’s 59 end is required. The critical role played by
the ‘‘seed sequence’’ in the majority of miRNA/mRNA
interactions implies that a single nucleotide change in the
seed sequence, or shift of the processing sites during
biogenesis of the miRNA/miRNA* duplex could result in
a novel miRNA with alternated target-spectra. Therefore,
both the 59 end of the mature miRNA that is generated
from the 59 arm of the pre-miRNA (5p) by Drosha, and the
59 end of the mature miRNA that is produced by Dicer
from the 39 arm of the pre-miRNA (3p), will be under
strong selective pressure to be highly conserved. The
sequence preceding the 59 end or trailing the 39 end of the
pre-miRNAs form an z11-base-pair (bp)-long imperfect
stem that is recognized by DGCR8 as part of the required
structure for Drosha cutting (Han et al. 2004, 2006). The
terminal loop is also important for Dicer/TRBP complex
binding (Zeng 2006), as well as for other protein binding
(Viswanathan et al. 2008). Sequences outside of the seed in
the mature miRNA sequence can also impact the strength of
inhibition as well as the spectra of targeted transcripts.
The hairpin structure-guided miRNA processing, the
thermodynamic influences on strand loading, and the
base-pairing requirements for miRNA/mRNA interaction
imply that single nucleotide polymorphisms (SNPs) in
miRNA genes should affect miRNA biogenesis and func-
tion. Similarly, SNPs in the miRNA target also affect
miRNA function. To clarify possible confusions in termi-
nology with respect to SNPs, we use the terms ‘‘miR-SNP’’
to refer to the variation that occurs in the miRNA gene
sequence, and ‘‘miR-TS-SNP’’ to refer to SNPs that occur
in the miRNA target site (TS) or binding site. Since one
miRNA can have multiple targets, miR-SNPs would be
expected to exhibit more profound and broader biological
effects than miR-TS-SNPs.
The roles that sequences flanking the pre-miRNA play in
miRNA processing have been thoroughly studied (Han
et al. 2004; Zeng an Cullen 2004, 2005; Zeng et al. 2005;
Han et al. 2006). miR-SNPs in miR-125a and Kaposi’s
sarcoma-associated herpes virus-encoded miR-K5 were
reported to impair miRNA processing by the Drosha/
DGCR8 complex (Gottwein et al. 2006; Duan et al.
2007). miR-196a2-SNP (rs11614913) in the mature miR-
196a2 was reported to be associated with a significantly
decreased rate of survival in individuals with non-small cell
lung cancer, and the investigators of this study also
suggested an association of rs11614913 with enhanced
processing of mature miR-196a (Hu et al. 2008a). miR-
146a-SNP (rs2910164) within the pre-miR-146a sequence
reduced both the amount of pre- and mature miR-146a,
and apparently affected the Drosha/DGCR8 processing step
(Hu et al. 2008b; Jazdzewski et al. 2008; Shen et al. 2008).
miR-196a2-SNP, miR-146a-SNP, miR-149-SNP (rs2292832),
and miR-499-SNP (rs3746444) are each associated with
increased breast cancer risk (Hu et al. 2008b). miR-146a-
SNP was associated with papillary thyroid carcinoma
(Jazdzewski et al. 2008), breast/ovarian cancer (Shen et al.
2008), and hepatocellular carcinoma (Xu et al. 2008).
The miR-146a-SNP and hepatocellular carcinoma report
is somewhat controversial since it claims that the SNP
enhanced miR-146a expression, while three other reports
show reduced expression. Each of the above are examples
of SNPs created by changes in DNA-coding sequences, but
miRNAs can also be post-transcriptionally modified, such
as by RNA editing via ADAR. A to I-edited pre-miR-151
blocks its processing by Dicer/TRBP (Kawahara et al.
2007a). ADAR-edited pri-miR-142 was more easily de-
graded by Tudor-SN (Yang et al. 2006). Edited miR-376a-
5p within the middle of the ‘‘seed’’ region alters the set of
targets regulated by this miRNA (Kawahara et al. 2007b). A
survey of RNA editing of miRNAs from 10 human tissues
implies RNA editing of miRNA happens quite often and it
is a mechanism to increase the diversity of miRNAs and
their targets (Blow et al. 2006).
The above examples show that mutant or post-transcrip-
tionally edited miRNAs can result in alterations of process-
ing and function. Hence, SNPs that occur in sequences
downstream from or upstream of the pre-miRNA, sequences
SNPs in human miRNA genes
in the terminal loop of pre-miRNA, and sequences in the
miRNA and miRNA* duplexes may also play important
roles in miRNA biogenesis and function (Supplemental Fig.
We have been analyzing X-linked miRNA genes from
male patients with diagnosed schizophrenia or autism, and
compared our findings with a gene pool analysis consisting
of over 7000 chromosomes from normal individuals. The
statistical correlation of SNPs in miRNAs with Schizophre-
nia is presented in a separate study (Feng et al. 2009). The
present study represents the first large-scale, systematic
genetic analysis of naturally occurring miR-SNPs. Twenty-
four different point mutations have been determined in
either the mature miRNA sequences or the precursor
regions for sixteen different X-linked miRNA genes. Here,
we address the effects on miRNA generation and function
generated by SNPs in X-linked miRNAs.
Of the tested miR-SNPs, one variant resulted in elevated
levels of the mature miRNA sequence, several variants
resulted in reduced levels of the mature miRNA sequence
relative to wild type, and another variant resulted in the
generation of a novel miRNA due to an alteration in the
Drosha and/or Dicer processing sites. This latter miRNA-
SNP also had an alteration in the strand-loading bias
relative to the wild-type version. Our results demonstrate
that a single base alteration even outside of the mature
miRNA sequence can have profound consequences on
miRNA generation and function.
A total of 59 microRNA genes on the X chromosome
(covered in miRBase 8–10 when the experiments were
conducted; the other 18 X-linked miRNAs in the current
miRBase 12.0 were not available at the time of this study)
were analyzed in 193 male schizophrenia patients and 191
male controls. Some variants were further screened by
analyzing whether or not they were present in 7000 control
chromosomes from normal individuals.
Twenty-four variants within pre-miRNAs and the imme-
diate flanking regions were identified (Table 1). They are
further characterized into four groups based on the location
within the hairpin structure (Supplemental Fig. S1).
In order to evaluate the consequences of these point
mutations, we have developed a facile assay to monitor the
processing and function of both strands of the miRNAs by
using both miRNA (seed sequence complementarity) and
siRNA (fully complementary) assays. The functional assays
were carried out in transient cotransfections of expressed
pri-miRNA with target sequences in the 39UTR of the
Renilla luciferase encoding transcripts. At least six of the
variants, miR-502-C/G (Fig. 1A, rare variant and associated
with schizophrenia), miR-510-T/C (Fig. 2A, rare variant
and associated with schizophrenia), miR-510-G/A (Fig.
2C), miR-890-C/G (Fig. 3A), miR-892b-T/C (Fig. 4A),
and miR-934-T/G (Fig. 5A), each showed reduced or
enhanced repression of the ‘‘si’’ and ‘‘mi’’ reporters in
transient transfection assays. For each of these variants,
Northern blotting was performed to detect the effects of the
SNP on processing of the pre- and mature miRNAs.
Mature miRNA cloning was performed on miR-510-G/A,
miR-890-C/G, and miR-934-T/G to study the affect of the
SNP on the maturation of the miRNAs. While all three
SNPs apparently affect the miRNA processing, we observed
that only the miR-934-T/G transversion also altered the
Drosha or Dicer excision sites (Supplemental Table S1),
which also resulted in changing the strand bias for RISC
loading relative to the wild-type miRNA (Fig. 5).
Variant miRNAs impair miRNA processing
In our tests, we observed several examples in which miR-
SNPs resulted in reduced processing. Four of the six
observed miR-SNPs, miR-502-C/G, miR-510-T/C, miR-
890-C/G, and miR-892b-T/C, produced less-detectable
mature miRNAs than the wild-type versions of these
miRNAs. While the 502-G/C SNP occurs 2 nt before the
59 end of 502-5p, the other three SNPs all occur in the
mature 3p products.
TABLE 1. List of all miRNA gene variants that were found in the
control population or the patient samplesa
5p 11: G>A
Stem–loop 32: A>G
3p 60: C>T
Stem–loop 41: G>A
Stem–loop 66/3p 6: G>A
Stem–loop 73: G>A
4 nt downstream from the 39
end of the stem–loop: G>A
5p 4: T>C
Stem–loop 13: C>G
Stem–loop 8: C>T
Stem–loop 54: ins TGA
Stem–loop 9: G>T
5p 11: ‘‘A’’ deletion
5p 22: G>A
5p 19: C>G
3p 13: C>T
Stem–loop 48/3p 4: T>C
Stem–loop 6: G>A
5p 15: C>T
Stem–loop 77: A>C
Stem–loop 66: G>C
Stem–loop 35: C>G
3p 15: T>C
5p 1: T>G
aRefer to Supplemental Fig. S2 for detailed SNP.
Sun et al.
RNA, Vol. 15, No. 9
Variant miR-502-C/G: This variant produces a bulge that
changes the structure of the stem of the precursor miRNA
(Fig. 1A). Most likely, this structural change affects the site
of Drosha cleavage in producing pre-miR-502. Reduced
target knockdowns were observed in the transfection assays
(Fig. 1B). The impaired functional activity of the variant
was supported by Northern blot analysis, as the production
of pre-miR-502 and mature 502-5p/3p was both reduced
Variant miR-510-T/C: Currently there are no published
reports to validate the informatic prediction of the miR-
510-3p products. Our 3p reporter transfection assays show
its ability to knockdown the corresponding ‘‘si’’ target
sequence (Fig. 2B). The T/C transition produces a pre-miR-
510 with much less activity for both 5p and 3p products
(Fig. 2B). Therefore, this mutation affects the function of
both strands of mature miR-510. Northern blot analyses
confirmed that the production of both pre-miR-510 and
miR-510-5p/3p are reduced (Fig. 2E).
Variant miR-890-C/G: Currently there are no published
data to validate the predicted miR-890 3p products. Our
3p reporter transfection assays show its ability to knock-
down the corresponding ‘‘si’’ target sequence. Transfection
and Northern blotting data show that the C/G transversion
in miR-890 affects the production of both the 3p and the
5p strands (Fig. 3B,C) with the production of both strands
being reduced. Because the C/G transversion may be at the
Drosha cleavage site, we wanted to define the exact
sequence of its 3p products by miRNA cloning. The clon-
ing data show that the cutting sites for both the 5p and
3p products were not altered by this miR-SNP (Supple-
mental Table S1). The 5p and 3p mature sequences in the
cloning data were the same for both the wild-type and
mutant. Dot blotting analyses (Supplemental Fig. S4b) also
show that more clones of the miR-890 than miR-890-C/G
for both the 3p and 5p probes were obtained, which is
consistent with the transfection and Northern blot data
FIGURE 1. Functional test of miR-502 and miR-502-C/G. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-502, miR-502-
C/G, and the sequences inserted into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (B) Cotransfection test results. The knockdown of 5p-si,
5p-mi, and 3p-si reporters is reduced in the mutant, whereas the expression of 3p-mi reporter is the same in the WT and Mutant. Each bar
represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD.
(C) Northern blot results. Blot was hybridized with miR-502-3p probe (right) and a miR-502-5p probe (left). (Lane 1) RNAs from cells transfected
with the WT miRNA construct; (lane 2) samples from cells transfected with the Mutant miRNA construct; (lane 3) RNAs from cells transfected
with the miRNA expression vector fU1-miR. U2 and U6 snoRNA was used as an RNA loading control. Quantification of the signal was first
normalized to U2, and then it was further normalized to the band with the lowest signal.
SNPs in human miRNA genes
Variant miR-892b-T/C: like miR-510-T/C and miR-890-
C/G, this SNP occurs in the 3p of miR-892b, but miR-
892b-3p is listed in miRBase. Although there are currently
no published data that support the existence of miR-892b-
5p products, our 5p reporter transfection assays show its
ability to knockdown the corresponding ‘‘si’’ target
FIGURE 2. (Continued on next page)
Sun et al.
RNA, Vol. 15, No. 9
sequence. Transfection and Northern blotting data show
that the T/C transition in miR-892b affects the production
of both 5p and 3p strands (Fig. 4B,C).
Variants in miRNA that enhance processing of mature
We were surprised to find that a G/A transition in pri-miR-
510 enhanced the production of miR-510-5p and -3p (-3p
is miR-510*) (Fig. 2D,E). The G-to-A transition occurs at
the fourth nucleotide upstream of the 59 end of the mature
miR-510-5p (Fig. 2C). Variants at this position may affect
Drosha processing of this substrate, since it may provide a
more stable stem preceding the mature miRNA sequence.
The reporter assay data show that the siRNA activity of the
mutant is markedly higher than the wild type (Fig. 2D).
Northern blot data show that the production of both pre-
miR-510 and mature miR-510-5p/3p are increased (Fig.
2E). Dot-blotting data also revealed more clones of the
miR-510-G/A than miR-510 (Supplemental Fig. S4A). The
miRNA cloning data show that the generation of the 5p
product is the same for both the wild type and mutant, and
apparently this SNP does not affect the Drosha cutting sites
(Supplemental Table S1). However, we didn’t observe any
FIGURE 2. Functional test of miR-510, miR-510-T/C, and miR-510-G/A. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-
510, miR-510-T/C, and the sequences inserted into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (B) Transfection test results of T/C mutant.
The knockdown of reporters for 5p-si, 5p-mi, 3p-si, and 3p-m-si (mutant form) from the mutant form are reduced. The repression for 3p-mi is
approximately the same for both the WT and the mutant. Each bar represents the average of at least three independent transfections with
duplicate determinations for each construct. Error bars represent the SD. (C) Sequences of miRNAs and reporters. Stem–loop sequences of miR-
510, miR-510-G/A, and the inserted sequences into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (D) Transfection test results of miR-510-
G/A. The knockdown of reporters for 5p-si, 5p-mi, 3p-si, and 3p-m-si (G/A mutant form) from the mutant are all increased. Each bar represents
the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (E) Northern
blot results. Blot was probed with the miR-510-5p probe (right) and miR-510-3p probe (left); U2 and U6 snoRNAs were used as RNA sample
loading controls. (Lane 1) Transfected with fU1-miR; (lane 2) transfected with miR-510 WT; (lane 3) transfected with the miR-510-T/C mutant;
(lane 4) transfected with fU1-miR-510-G/A. Quantification of the signal was first normalized to U2, and then it was further normalized to the
band with the lowest signal.
SNPs in human miRNA genes
colonies hybridizing to the 3p probe in either the wild-type
or SNP blots; therefore, the exact sequence of miR-510-3p
Variant miRNA with altered mature miRNA
processing sites and miRISC strand loading bias
It is reasonable that SNPs could alternate Drosha or Dicer
excision sites, since their cutting sites are structure based
and not sequence based. Variant miR-934-T/G occurs at
the first nucleotide of the miR-934-5p (Fig. 5A), which is
also the Drosha processing site. Because the variant occurs
at the 59 end and the base of the 59end plays an important
role in strand selection into miRISC, the T/G transversion
of this variant is particularly interesting. Transfection and
Northern blot results show that this SNP affects the
production of both strands (Fig. 5B,C). First, the trans-
fection assay shows that repression of the 5p reporter is
reduced by the SNP, and Northern blots confirmed the
reporter assay results (Fig. 5B). Second, the length of the 5p
product seems to be increased in the Northern blot. The
most dramatic changes are in the 3p product. Transfections
show that repression of the 3p reporter by the SNP is
increased, and Northern blots show that the variant
produces more 3p than wild type. Thus, the guide strand
and passenger strand in miRISC are inverted in the wild-
type versus mutant miRNAs. Cloning also yielded more 5p
wild-type clones and more mutant 3p clones (Supplemental
Fig. S4c). The cloning data also show that the production of
3p is altered, with both the Drosha and Dicer cutting sites
being offset by one nucleotide from the wild type, resulting
in a different 3p product (Supplemental Table S1). This
not only produced a novel miRNA, but it also affected
the strand selection in miR-934/miR-934*. The wild-type
miR-934-5p starts with a U and is most likely selected
as the predominant guide strand due to the lower thermo-
dynamic stability of the 59 end. The U/G transversion
changes the first nucleotide of the 5p product to ‘‘G<’’
FIGURE 3. Functional test of miR-890 and miR-890-G/C. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-890, miR-890-
G/C, and the sequences inserted into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (B) Transfection test results. The knockdown of reporters
for 5p-si, 5p-mi, 3p-si, and 3pGC-si (mutant form) from the mutant form are reduced. The expression of the 3p-mi reporter is approximately the
same for both the WT and mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for
each construct. Error bars represent the SD. (C) Northern blot results. Blot was probed with miR-890-5p, 3pGC, and 3p for samples transfected
with fU1-miR (lane 1), fU1-miR-890 (lane 2), and fU1-miR-890-G/C (lane 3). U2 and U6 snoRNAs were used as RNA loading controls.
Sun et al.
RNA, Vol. 15, No. 9
which affected the Dicer cutting site, moving it back one
nucleotide from the original ‘‘G’’ to an ‘‘A.’’ Thus, the 3p
product in the mutant has a lower 59 end thermodynamic
stability, and this is probably responsible for altered guide
strand selectivity, which is consistent with the reported
requirements for asymmetric strand loading (Schwarz et al.
miRNAs play an essential role in various biological func-
tions including development, cell differentiation, prolifer-
ation, viral pathogenesis, and progression of human
diseases (Bartel 2004; Bushati and Cohen 2007). Spatial
and temporal expression of individual miRNAs, plus the
synergistic effects of miRNAs that bind to the same target,
make miRNAs a family of regulatory factors that can subtly
and precisely modulate target gene expression. The func-
tion of miRNAs can be compromised by variations in their
sequences (Gottwein et al. 2006; Duan et al. 2007) or their
target-site sequences (Kawahara et al. 2007b; Saunders et al.
2007). Our data are consistent with other published reports
which show that there are more SNPs that impair miRNA
generation than SNPs that enhance miRNA generation. It is
of interest that some miR-SNP-related reports did not
observe obvious aberrant processing of miR-SNPs, perhaps
owing to the subtle changes that such SNPs can have on the
processing reactions (Diederichs and Haber 2006; Chen
et al. 2007). In contrast, changes in miRNA-binding sites
(miR-TS-SNPs) are readily associated with loss of miRNA
function (Abelson et al. 2005; Clop et al. 2006; Arisawa
et al. 2007; Martin et al. 2007; Mishra et al. 2007;
Sethupathy et al. 2007; Yu et al. 2007).
miR-SNPs may contribute to the evolution of miRNAs
with new or altered functions. miR-SNPs may reveal a
mechanism for the generation of clustered miRNAs, miRNA
homologs, or family members of miRNAs during evolution.
Most miR-SNPs we observed are located in clusters and
some of them, such as mir-510 and miR-509, rapidly evolved
in primates (Zhang et al. 2007). One clear example is the
generation of miR-509-3-5p by the deletion of an ‘‘A’’ from
miR-509-5p, which is processed from miR-509-1 or 2
FIGURE 4. Functional test of miR-892b and miR-892b-T/C. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-892b, miR-
892b-T/C, and the sequences inserted into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (B) Transfection test results. The knockdown of
reporters for 5p-si, 3p-si, 3pm-si (mutant form), and 3p-mi from the mutant form are reduced. Each bar represents the average of at least three
independent transfections with duplicate determinations for each construct. Error bars represent the SD. (C) Northern blot results. Blot was
hybridized with probes for miR-892b-5p, 3p, and 3pTC (mutant form) using samples transfected with fU1-miR (lane 1), fU1-miR-892b (lane 2),
and fU1-miR-892b-T/C (lane 3). U2 and U6 snoRNAs were used as RNA loading controls.
SNPs in human miRNA genes
(Supplemental Fig. S2). There are three copies of miR-509,
miR-509-1, and miR-509-2 that produce the same mature
miRNAs, while miR-509-3 produces a different 5p product.
Most likely, the miR-509-3-5p was created by the deletion
of an ‘‘A’’ from miR-509-5p. We also observed a high
percentage of an ATG insertion in the 59 end of miR-509-1-
3p. This insertion may affect both 5p and 3p processing.
Eventually, under selective pressure to target different
mRNAs or to target with different specificities, the three
copies of miR-509 may have developed into different family
members with the same seed, like the let-7 family, or
different miRNAs in the same cluster, like the miR-25-93-
106b cluster. It appears to us that the miR-509 structure is
more flexible, since we did not observe significant differ-
ences in the processing or function among three different
miR-509-3 variants (Supplemental Fig. S3).
Many factors may contribute to differences in miRNA
expression profiles, including transcriptional regulation,
post-transcriptional miRNA processing, the stability of
the pri-miRNA or pre-miRNA, and pre-miRNA export.
The existence of miRNA targets may also result in miRNA
stabilization because of engagement in miRISC. Our data
clearly show different miRNA profiles as a consequence of
subtle genetic changes in pre-miRNAs and their immediate
Previous in silico studies from Bentwich et al. (2005) and
Zhang et al. (2007) show that miRNA family expansion
during primate evolution may have occurred through
tandem duplication. Copy-number variations and high
rates of gene conversion in the newly emerged miRNAs
in primates may have resulted in production of novel
miRNAs with more specialized functions. As a result, gene
conversion may be a major mechanism in the biogenesis of
miRNAs during evolution, especially in clusters of mi-
RNAs, homologs, or miRNA families. Finally, some of the
SNPs that we have characterized by altered processing or
abundance may play significant roles in disease develop-
ment and progression.
FIGURE 5. Functional test of miR-934 and miR-934-T/G. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-934, miR-934-
T/G, and the sequences inserted into the 39UTR of Rluc for the ‘‘si’’ and ‘‘mi’’ reporters. (B) Transfection test results. The knockdown of 5p-si and
5p-mi reporters by the mutant were reduced and strong knockdown of the 3p-si and 3pm2-si reporters are observed from the mutant miRNA.
Each bar represents the average of at least three independent transfections, with duplicate determinations for each construct. Error bars represent
the SD. (C) Northern blot results. Blot was probed with a miR-934-5p probe (left) and 3p probe (right). U2 and U6 snoRNAs were probed as RNA
gel loading controls. (Lanes 1,2) From cells transfected with fU1-miR; (lane 3) from cells transfected with fU1-miR-934; (lane 4) from cells
transfected with the fU1-miR-934-T/G.
Sun et al.
RNA, Vol. 15, No. 9
MATERIALS AND METHODS
Cell lines and plasmids
HEK293 cells were purchased from ATCC and were maintained in
high glucose (4.5 g/L) DMEM supplemented with 2 mM gluta-
mine, 10% FBS, and 2 mM Penicillin/Streptomycin. Transfections
to HEK293 cells were performed with Lipofectamine 2000
(Invitrogen) in duplicate in 24-well plate formats with cells at
Cell-based miRNA processing test
Primary miRNA expression plasmids and reporter constructs with
either fully complementary targets or seed sequences complemen-
tary to the miRNAs were cotransfected into HEK293 cells. Dual-
reporters (expressing both Firefly Luciferase [Fluc] and Renilla
Luciferase [Rluc]) carrying the fully complementary miRNA
targets (‘‘si’’ reporters) in the 39UTR of the Renilla transcript
were constructed. These were used to validate the expression level
and the ability of cloned primary miRNA expression plasmids to
produce functional, mature miRNAs. Dual-reporters carrying the
partially complementary sequence (‘‘mi’’ reporters: sequences that
are complementary to the mature miRNA sequence except for
mismatches at positions 11–13 and the last two nucleotides in the
miRNA/mRNA duplex) of a miRNA in the Rluc 39UTR were used
to quantitatively measure the translational repression from the
In order to express the pri-miRNAs, we retrieved the predicted
stem–loop sequences from miRBase (Griffiths-Jones et al. 2008).
The stem–loop sequence plus flanking sequences extending at least
100 bases in both directions were PCR amplified from patient
genomic DNAs when possible. An miRNA expression vector was
constructed by cloning the human Pol II U1 promoter upstream
of a multiple cloning site in the Bluescript SK plasmid to create
the plasmid SK-U1. Secondly, the U1 transcriptional termination
sequence was cloned downstream of the MCS of SK-U1 to create
the fU1-miR miRNA expression vector. The primary miRNA
sequence was cloned into the XhoI and BamHI sites of fU1-miR.
miRNA variants were cloned in the same manner as the wild-type
miRNAs from sample DNA when available. If samples were no
longer available, the QuikChange Site-Directed Mutagenesis Kit II
(Stratagene) was used to create mutants within the wild-type
All miRNAs and their homologous mutant ‘‘si/mi’’ reporters
were generated by inserting annealed oligos into the psiCheck2.2
XhoI/SpeI sites or XhoI/NotI sites in the 39UTR of the Rluc.
About 5 3 104HEK293 cells per well in 500 mL of growth
media were plated in 24-well plates 1 d prior to transfection. The
cells were at 80% confluency at the time of transfection. Each well
was transfected with 5 ng of reporter, 100 ng of miRNA expression
constructs (1:20 reporter/miRNA ratio; 1:5, 1:1 or 5:1 ratio was
used if the knock down of the ‘‘si’’ target was very efficient, e.g.,
>95%: 1:5 as 25 ng of miRNA expression plasmid and 75 ng of
stuffer Blue-script SK) and 1 uL of Lipofectomine 2000. Forty
eight hours post transfection, the cells were lysed with 100 mL of
Passive Lysis Buffer (Promega) and Luciferase levels were analyzed
from 20 mL of lysates using the Dual Luciferase reporter assay (50
mL of each substrate reagent, Promega) on a Veritas Microplate
Luminometer (Turner Biosystems). Changes in expression of Rluc
(target) were calculated relative to Fluc (internal control) and
normalized to the miRNA expression vector control (fU1-miR).
Mutagenesis of miRNA target sites
When the DNA from patient samples containing the SNPs was
not available, the appropriate changes were introduced into the
wild-type sequences using the QuikChange Site-Directed Muta-
genesis Kit II (Stratagene) following the protocol provided in the
kit. Mutations were confirmed by sequencing.
RNA isolation, Northern blot, and mature miRNA
RNA isolation, Northern blot, and small RNA cloning were
carried out as previously reported (Sun et al. 2007). Briefly, RNA
was isolated with RNA STAT-60 (Tel-Test, Inc.) and 20 mg of total
RNA was loaded into a denaturing 12.5% SDS–polyacrylamide
gel. A DNA oligonucleotide probe complementary to the mature
miRNA sequence was labeled with [g-32P]ATP. For Northern
blots, at least two independent transfections with different prepa-
ration of plasmids were performed in HEK293 cells to detect
processing of expressed pri-miRNAs. One transfection contained
pri-miRNA expression constructs alone, while the other one was
cotransfected with a control 25/27-mer dicer substrate siRNA that
targets HIV Tat/Rev as transfection efficiency control (only one
set of the data was showed in the figures). The hybridization was
performed overnight in PerfectHyb Plus Hybridization Buffer
from Sigma. The blots were washed once for 10–30 min with
6x SSPE/0.1% SDS, followed by two washings with 6x SSC/0.1%
SDS for 10–30 min each. U2 or U6 snoRNA were used as RNA
loading controls. For small RNA cloning, small RNAs below 40 nt
were fractionized by a flashPAGE Fractionator System. Small
RNAs were first polyadenylated, then ligated with a 59 RNA adap-
tor. The 59-adaptor-added polyadenylated small RNAs were RT-
PCR amplified and the products cloned. Dot-blot hybridizations
were used to identify positive clones. The positively hybridizing
clones were sequenced to verify the processed mature miRNA
Bio-Rad membranes were cut to the same size as the bottom of
Petri-Dish plates. The membranes were laid on the colonies for 20
sec or until they were wet, then lifted and washed twice in 0.5N
NaOH for 5 min each (the plates were put back into the 37°C
incubator for 5–6 h to preserve the colonies). Next, the mem-
branes were washed twice in 0.5M Tris-HCl (pH 7.5) for 5 min
each. Then, the membranes were washed twice in 6x SSC/0.1%
SDS for 5 min each. Finally, the membranes were washed in 95%
EtOH for 5 min and dried between two sheets of Whatman paper.
All washings were performed at room temperature. Just before
hybridization, membranes were soaked in 6x SSPE/0.1% SDS
twice for 5 min each. The probe and the temperature of
hybridization and the washing condition were the same as those
for the Northern blots above. The only difference was that the
hybridization duration was 1 h. Usually the signal is strong
enough to detect after the blots are exposed to film for 5–6 h.
Positive colonies were located and plasmid DNAs were made for
SNPs in human miRNA genes
Supplemental material can be found at http://www.rnajournal.org.
This work was supported by NIH grants AI29329 and HL07470 to
J.J.R. We wish to thank Brain Luk for critical reading of this
Received January 14, 2009; accepted May 26, 2009.
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