Correlation between sequence conservation and structural thermodynamics of microRNA precursors from human, mouse, and chicken genomes.

Abstract

Previous studies have shown that microRNA precursors (pre-miRNAs) have considerably more stable secondary structures than other native RNAs (tRNA, rRNA, and mRNA) and artificial RNA sequences. However, pre-miRNAs with ultra stable secondary structures have not been investigated. It is not known if there is a tendency in pre-miRNA sequences towards or against ultra stable structures? Furthermore, the relationship between the structural thermodynamic stability of pre-miRNA and their evolution remains unclear.
We investigated the correlation between pre-miRNA sequence conservation and structural stability as measured by adjusted minimum folding free energies in pre-miRNAs isolated from human, mouse, and chicken. The analysis revealed that conserved and non-conserved pre-miRNA sequences had structures with similar average stabilities. However, the relatively ultra stable and unstable pre-miRNAs were more likely to be non-conserved than pre-miRNAs with moderate stability. Non-conserved pre-miRNAs had more G+C than A+U nucleotides, while conserved pre-miRNAs contained more A+U nucleotides. Notably, the U content of conserved pre-miRNAs was especially higher than that of non-conserved pre-miRNAs. Further investigations showed that conserved and non-conserved pre-miRNAs exhibited different structural element features, even though they had comparable levels of stability.
We proposed that there is a correlation between structural thermodynamic stability and sequence conservation for pre-miRNAs from human, mouse, and chicken genomes. Our analyses suggested that pre-miRNAs with relatively ultra stable or unstable structures were less favoured by natural selection than those with moderately stable structures. Comparison of nucleotide compositions between non-conserved and conserved pre-miRNAs indicated the importance of U nucleotides in the pre-miRNA evolutionary process. Several characteristic structural elements were also detected in conserved pre-miRNAs.

Full text

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RESEARCH ARTICLEOpen Access
Correlation between sequence conservation and
structural thermodynamics of microRNA
precursors from human, mouse,
and chicken genomes
Ming Ni1,2, Wenjie Shu2, Xiaochen Bo2, Shengqi Wang2*, Songgang Li1*
Abstract
Background: Previous studies have shown that microRNA precursors (pre-miRNAs) have considerably more stable
secondary structures than other native RNAs (tRNA, rRNA, and mRNA) and artificial RNA sequences. However,
pre-miRNAs with ultra stable secondary structures have not been investigated. It is not known if there is a
tendency in pre-miRNA sequences towards or against ultra stable structures? Furthermore, the relationship
between the structural thermodynamic stability of pre-miRNA and their evolution remains unclear.
Results: We investigated the correlation between pre-miRNA sequence conservation and structural stability as
measured by adjusted minimum folding free energies in pre-miRNAs isolated from human, mouse, and chicken.
The analysis revealed that conserved and non-conserved pre-miRNA sequences had structures with similar average
stabilities. However, the relatively ultra stable and unstable pre-miRNAs were more likely to be non-conserved than
pre-miRNAs with moderate stability. Non-conserved pre-miRNAs had more G+C than A+U nucleotides, while
conserved pre-miRNAs contained more A+U nucleotides. Notably, the U content of conserved pre-miRNAs was
especially higher than that of non-conserved pre-miRNAs. Further investigations showed that conserved and
non-conserved pre-miRNAs exhibited different structural element features, even though they had comparable
levels of stability.
Conclusions: We proposed that there is a correlation between structural thermodynamic stability and sequence
conservation for pre-miRNAs from human, mouse, and chicken genomes. Our analyses suggested that pre-miRNAs
with relatively ultra stable or unstable structures were less favoured by natural selection than those with
moderately stable structures. Comparison of nucleotide compositions between non-conserved and conserved
pre-miRNAs indicated the importance of U nucleotides in the pre-miRNA evolutionary process. Several
characteristic structural elements were also detected in conserved pre-miRNAs.
Background
MicroRNAs (miRNAs) are small endogenous non-cod-
ing RNAs that regulate expression at the post-transcrip-
tional level in animals and plants [1]. Both plant and
animal miRNAs are cleaved from one arm of foldback
precursors (pre-miRNAs). It is generally accepted that
pre-miRNA secondary and/or tertiary structures are cri-
tical in miRNA biogenesis [2-5]. The thermodynamic
stability of pre-miRNA hairpin secondary structures,
hereafter called pre-miRNA stability, is a fundamental
property of RNA structure and has been systematically
studied. Bonnet et al. reported that in five animal spe-
cies and one plant species pre-miRNAs have signifi-
cantly lower estimated folding minimum free energies
(MFEs) than those of their shuffled sequences, unlike
other kinds of RNAs such as tRNAs, rRNAs [6], and
mRNAs [6,7]. Zhang et al. directly compared the stabi-
lity of pre-miRNAs and other kinds of RNAs in seven
* Correspondence: sqwang@bmi.ac.cn; lsg@pku.edu.cn
1Center for Bioinformatics, National Laboratory of Protein Engineering and
Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing
100871, China
2Beijing Institute of Radiation Medicine, Beijing 100850, China
Full list of author information is available at the end of the article
Ni et al. BMC Evolutionary Biology 2010, 10:329
http://www.biomedcentral.com/1471-2148/10/329
© 2010 Ni et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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plant species and showed that pre-miRNAs form more
stable secondary structures [8]. Currently, the lower
limit of pre-miRNA thermodynamic stability is widely
used as a criterion for predicting and verifying
sequences of RNA that constitute pre-miRNA [9-12].
However, several studies have characterized pre-
miRNA sequence and structural features that can lead
to pre-miRNA instability. The unwinding of pre-miRNA
foldback duplex structure is critical for processing of
pre-miRNAs [13,14]. Therefore, less stable pre-miRNAs
may be processed more easily. Pre-miRNAs have higher
total adenine (A) and uracil (U) contents than other
kinds of RNAs [8]. As A-U and guanine (G)-cytosine
(C) form two and three hydrogen bonds respectively,
pre-miRNAs with a higher A+U content may be less
stable. Some studies of miRNA biogenesis in animal
species have suggested that instability, or enhanced flex-
ibility of pre-miRNAs, resulting from mismatched
nucleotides, bulges, and especially unstable base pairs at
the 5’ end, can increase the efficiency of Dicer enzymes
involved miRNA biogenesis [15-17]. The human nuclear
processing enzyme Drosha has also been found to selec-
tively cleave pre-miRNAs hairpins bearing a large term-
inal loop (≥ 10 nucleotides) [18]. This type of terminal
loop could also result in pre-miRNA instability. While
these studies provided evidence for structural and
sequence features that destabilize pre-miRNAs, a sys-
tematic investigation of pre-miRNA instability has not
been carried out. It has been shown that there is a ten-
dency against unstable pre-miRNA structures [6,8].
However, the question remains: is there a tendency
against ultra stable secondary structures in pre-miRNA
sequences?
A correlation between pre-miRNA stability and
nucleotide sequence conservation would be expected
due to natural selection, if there is a range of stability
that directly or indirectly results in efficient pre-miRNA
functioning. MiRNAs were previously regarded as highly
conserved [1], but a number of non-conserved miRNAs
have been recently found in closely related species
[1,9,19-23]. To investigate the relationship between
sequence conservation and thermodynamic stability, we
compared the thermodynamic stability of conserved and
non-conserved pre-miRNAs from human, mouse, and
chicken genomes, with special emphasis on ultra stable
and unstable pre-miRNA sequences. We also investi-
gated the correlation between pre-miRNA structural ele-
ments and sequence conservation.
Results
Stability comparison between conserved and non-
conserved pre-miRNAs
We calculated the minimum free energy (MFE) values of
658 conserved and non-conserved pre-miRNAs. As the
MFEs increased linearly with sequence length, we nor-
malized the AMFE values to 100 nucleotide sequence to
yield adjusted MFEs (AMFEs) [8] for comparing pre-
miRNA thermodynamic stability (Additional file 1).
Potential artefacts arising from difference sequence
lengths were excluded by using AMFEs for comparisons.
Conserved pre-miRNAs were divided into three conser-
vation levels, termed as Sc1, Sc2, and Sc3, representing
low, moderate, and high levels of conservation, respec-
tively (see Methods). Non-conserved pre-miRNAs were
classed as Sn. The summary of the pre-miRNA stability,
sequence length, and conservation was given in
Additional file 2_sheet 1 (human), 2 (mouse), and 3
(chicken). The distributions of AMFEs for the conserved
and the non-conserved pre-miRNAs are shown in
Figure 1. AMFEs in conserved and non-conserved pre-
miRNA groups were from normal distribution expect
mouse Sc1group (Additional file 3), and for each species
we statistically compared the mean AMFE and AMFE
variances of Snwith Sc1, Sc2, and Sc3(Table 1). For pre-
miRNAs from the human and chicken genomes, the
mean AMFEs of the non-conserved and conserved pre-
miRNAs were not statistically different. For pre-miRNAs
from the mouse genome, although the mean AMFEs of
Sc1and Sc2were significantly larger than that of Snat
the 0.05 FDR level, the mean AMFE difference
decreased and became non-significant for the Sc3pre-
miRNAs. The mean AMFE of Sc3was only 2.8 kcal/mol
higher than that of Sn, while the mean AMFE of Sc1was
4.1 kcal/mol higher. Therefore, although pre-miRNAs
are significantly more stable than other non-coding
RNAs [8], the conserved and non-conserved pre-
miRNAs had relatively similar mean AMFEs.
Unlike the mean AMFE values, the AMFE variances of
the conserved and non-conserved pre-miRNAs were sig-
nificantly different in all three species at the FDR 0.05
level (Table 1). Moreover, the AMFE variance consis-
tently decreased from Sc1to Sc3in all three species (in
chicken Sc1was excluded). The AMFE standard devia-
tions of Snwere 2.21-fold, 1.66-fold, and 2.91-fold larger
than Sc3for pre-miRNAs from the human, mouse, and
chicken genomes, respectively.
Conserved and non-conserved pre-miRNAs were
classed as relatively ultra stable or less stable to further
investigate the stability distribution of pre-miRNAs. In
each species, pre-miRNAs with AMFEs in the top 10
percent were classed as ultra stable, and in the bottom
10 percent were classed as unstable. In the Sc3group,
3.0% (human), 2.6% (mouse), and 0.0% (chicken) of pre-
miRNAs were ultra stable. In comparison, in the Sn
group, 16.7% (human), 9.3% (mouse), and 23.8%
(chicken) were ultra stable. Similar results were obtained
for unstable pre-miRNAs. In the Sc3group, 3.0%
(human), 2.6% (mouse), and 5.2% (chicken) of pre-
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miRNAs were unstable, while in the Sngroup, 18.2%
(human), 19.4% (mouse), and 23.8% (chicken) of pre-
miRNA were unstable. In summary, the mean AMFEs
of conserved and non-conserved pre-miRNA AMFEs
were similar, but the distribution of AMFEs for
conserved pre-miRNAs was significantly smaller than
that of non-conserved pre-miRNAs.
Sequence comparison between conserved and non-
conserved pre-miRNAs
We next investigated how the pre-miRNA nucleotide
compositions were correlated with sequence conserva-
tion. Firstly, for all pre-miRNAs, we calculated the pro-
portion of nucleotides forming base pairs (bp %) and
the proportion of these base pairs that were A-U base
pairs ((A-U) %) (Table 2, Additional file 4, see also
Additional file 3 for normality test results). On average,
the conserved pre-miRNAs contained significantly more
base pairs (72.4%) than the non-conserved pre-miRNAs
(68.3%, p = 8.7 × 10-14). However, the mean values of
(A-U) % for the conserved pre-miRNAs were also signif-
icantly larger than those of the non-conserved pre-miR-
NAs (at 0.01 FDR level, in human and chicken; at 0.05
FDR level, mouse). On average, 49.2% (human), 48.6%
(mouse), and 51.8% (chicken) base pairs of the con-
served pre-miRNAs were A-U, compared with 42.7%
(human), 44.4% (mouse), and 37.1% (chicken) of the
non-conserved pre-miRNAs. In this study, conserved
and non-conserved pre-miRNAs had similar mean
AMFEs. This is consistent with the base pairing results
as A-U base pairs are less stable than G-C base pairs.
So, although the conserved pre-miRNAs have a higher
bp % than the non-conserved pre-miRNAs, this is offset
by a larger proportion of A-U base pairs. On the other
hand, the distribution variances of both bp % and (A-U)
0
20
40
60
80
AMFE (kcal/mol)
Sn (132)
Sc1 (60)
Sc2 (66)
Sc3 (33)
Human
0
20
40
60
80
AMFE (kcal/mol)
Sn (108)
Sc1 (63)
Sc2 (75)
Sc3 (39)
Mouse
0
20
40
60
80
AMFE (kcal/mol)
Sn (21)
Sc1 (<5)
Sc2 (40)
Sc3 (19)
Chicken
A
B
C
Figure 1 Distribution of AMFEs. Boxplot of AMFEs for non-
conserved (Sn, red) and conserved pre-miRNAs (Sc1, Sc2, and Sc3,
black) from human (A), mouse (B), and chicken genomes (C). The
number of pre-miRNAs within each set is indicated in brackets. Sc1
for chicken was excluded as it contained fewer than five pre-
miRNAs.
Table 1 AMFE Comparison between non-conserved and
conserved pre-miRNAs from human, mouse, and chicken
genomes
SpeciesConservationAMFE
(kcal/mol)
p1
p2
Human
Sn(132)
Sc1(60)
Sc2(66)
Sc3(33)
Sn(108)
Sc1(63)
Sc2(75)
Sc3(39)
Sn(21)
Sc2(40)
Sc3(19)
44.1 ± 14.1
45.2 ± 9.98
44.6 ± 7.06
44.7 ± 6.36
5.4×10-1
7.8×10-1
7.5×10-1
*3.7×10-3
*8.6×10-9
*2.3×10-6
Mouse41.0 ± 9.79
45.1 ± 7.83
45.0 ± 6.93
43.8 ± 5.89
*5.2×10-3
*2.4×10-3
5.6×10-2
*4.5×10-2
*1.4×10-4
*4.8×10-4
Chickena
42.2 ± 14.7
41.6 ± 5.59
42.3 ± 5.05
8.7×10-1
9.8×10-1
*3.3×10-7
*3.1×10-5
The number of pre-miRNAs within each set is indicated in brackets behind set
name.aSc1for chicken was excluded as it contained fewer than five pre-
miRNAs. AMFE, the AMFE mean values (± SD). Pair-wise comparisons were
performed between non-conserved (Sn) and conserved pre-miRNAs (Sc1, Sc2,
and Sc3) from human, mouse, and chicken genomes. p1, P-value of two-
sample two-sided t-test (without assuming equal variance) for AMFE means.
p2, P-value of two-sample two-sided F-test for AMFE variances. *the P-value is
significant at the 0.05 FDR level.
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% could explain the observed stability variances. Con-
served pre-miRNAs had consistently smaller bp % and
(A-U) % variances than non-conserved pre-miRNAs.
The nucleotide composition of the conserved and
non-conserved pre-miRNAs was also examined, as
shown in Table 2 (see also Additional file 3 for normal-
ity test results). All conserved pre-miRNA sets had
higher average A+U contents than G+C contents, while
the A+U contents of the non-conserved pre-miRNA sets
were all below 50% and significantly smaller than the
conserved pre-miRNA sets, expect for the mouse Sc2set.
Compared with non-conserved pre-miRNAs, conserved
pre-miRNAs had an increase of A and U contents but a
decrease of G and C contents (Figure 2). Notably, the U
content of conserved pre-miRNA was higher than A, G,
and C. The average U content in overall conserved pre-
miRNAs was 29.4% while the average A content was
23.4%. Furthermore, conserved pre-miRNAs had 3.4%
higher U contents than non-conserved pre-miRNAs
while the A content was only 1.4% higher in conserved
pre-miRNAs than in non-conserved pre-miRNAs. In
contrast, the G and C contents were 3.0% and 2.1%
lower in conserved pre-miRNAs than in non-conserved
pre-miRNAs respectively. We also observed that the
nucleotide content of conserved pre-miRNAs has a nar-
rower distribution than non-conserved pre-miRNAs.
Structural element comparison between conserved and
non-conserved pre-miRNAs
Hairpin secondary structures can be divided into five
basic structural elements [24], including two kinds of
stems (interior and first stem) and three kinds of loops
(terminal, interior and overhang loops) (Figure 3A). We
examined whether conserved and non-conserved pre-
miRNAs differed with respect to structural elements.
We compared non-conserved pre-miRNAs (n = 260, Sn)
with the most conserved pre-miRNAs (n = 91, Sc3) from
the three genomes, focusing on two features: (1) the
ratio of the structural element length to the complete
sequence length and (2) the ratio of U nucleotides to
the total number of nucleotides within the structural
element. Two AMFE thresholds were selected (T1=
38.6 kcal/mol and T2= 48.6 kcal/mol) corresponding to
the 10thquantile and 90thAMFE quantile of the Sc3pre-
miRNAs respectively. Non-conserved pre-miRNAs were
denoted as Snu, Snm, and Snswith AMFEs <T1, T1≤
AMFEs ≤ T2, and AMFEs >T2, respectively. Scmdenotes
the conserved pre-miRNAs with AMFEs between T1
and T2(80% of the total conserved pre-miRNAs).
Although AMFEs for pre-miRNAs in the Scmand Snm
groups were comparable (Additional file 5), there was
considerable variation in structural elements (Figure
3B-F). For the non-conserved pre-miRNAs, interior
stem and first stem length ratios increased with increas-
ing sequence stability. Surprisingly, the Scmgroup exhib-
ited significantly larger interior stem ratios on average
than the Snsgroup (p = 2.5 × 10-5). The Snmgroup had
smaller overhangs than both the Snuand Snsgroups, but
the overhangs of the Scmgroup were even significantly
smaller than those of the Snmgroup (p = 3.2 × 10-4).
The Scmgroup also had significantly lower interior loop
ratios (p = 3.2 × 10-7) and larger terminal loop ratios
than the Snmgroup (p = 4.4 × 10-2). Only first stem
ratios of the Scmand Snmgroups were comparable. On
the other hand, the structural element U contents of
conserved and non-conserved pre-miRNAs are shown in
Figure 3G-J. The Scmgroup had significantly higher U
ratios at interior stem region (p = 3.1 × 10-3) than the
Snmgroup. The Scmgroup also had higher U content on
average than the Snugroup at terminal and interior
Table 2 Summary and comparison of pre-miRNA base pairing and nucleotide composition
SpeciesConservationbp % (A-U) %(A+U) %A %U %G % C %
Human
Sn(132)
Sc1(60)
Sc2(66)
Sc3(33)
Sn(108)
Sc1(63)
Sc2(75)
Sc3(39)
Sn(21)
Sc2(40)
Sc3(19)
68.66 ± 7.83
71.88 ± 6.20A
72.94 ± 5.64Ab
73.48 ± 4.67AB
42.73 ± 16.12
49.34 ± 11.81Ab
48.67 ± 8.17AB
50.14 ± 11.02Ab
47.11 ± 13.18
53.87 ± 10.66A
51.62 ± 7.15AB
52.94 ± 9.67a
22.10 ± 7.89
23.78 ± 6.36
23.41 ± 4.74B
22.96 ± 6.05
25.01 ± 6.96
30.09 ± 6.42A
28.21 ± 4.42AB
29.98 ± 6.16A
27.49 ± 7.90
24.45 ± 5.53AB
25.71 ± 3.49aB
26.06 ± 5.15B
25.40 ± 7.24
21.68 ± 5.84A
22.67 ± 5.11AB
20.99 ± 5.92A
Mouse 67.87 ± 6.51
72.53 ± 5.85A
72.10 ± 5.87A
73.37 ± 5.13A
44.35 ± 11.20
48.20 ± 11.47a
47.70 ± 8.14aB
50.85 ± 10.93A
49.30 ± 9.65
53.07 ± 9.42a
50.57 ± 6.77B
53.05 ± 9.15a
22.40 ± 6.16
23.29 ± 5.42
22.90 ± 4.57b
22.89 ± 5.90
26.90 ± 5.97
29.78 ± 6.11A
27.67 ± 4.40b
30.16 ± 5.51A
27.28 ± 5.57
25.30 ± 5.28a
26.28 ± 3.72B
26.06 ± 5.06
23.42 ± 5.74
21.62 ± 5.16a
23.15 ± 5.02
20.90 ± 5.53a
Chicken*68.24 ± 7.37
70.89 ± 7.06
73.08 ± 5.14a
37.13 ± 17.43
51.22 ± 7.19AB
53.80 ± 7.56AB
44.17 ± 14.75
55.11 ± 4.99AB
57.18 ± 5.72AB
19.47 ± 8.79
24.60 ± 4.14B
24.45 ± 5.26
24.69 ± 8.01
30.52 ± 3.97AB
32.74 ± 3.26AB
29.35 ± 6.00
24.80 ± 3.53AB
23.94 ± 4.49A
26.48 ± 9.74
20.08 ± 3.24aB
18.87 ± 3.33AB
Results represent the mean (± SD) of detailed information of base pairing and nucleotide composition. The number of pre-miRNAs within each set is indicated in
brackets behind set name. *Sc1for chicken was excluded as it contained fewer than five pre-miRNAs. Pair-wise comparisons of means (two-sided t-test) and
variances (F-test) were performed between non-conserved (Sn) and conserved pre-miRNAs (Sc1, Sc2, and Sc3) from human, mouse, and chicken genomes.amean
value significantly different from that of Snat the 0.05 FDR level.Amean value is significantly different from that of Snat the 0.01 FDR level.bvariance is
significantly different from that of Snat the 0.05 FDR level.Bvariance significantly different from that of Snat the 0.01 FDR level.
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loops, although the differences were not significant. No
apparent increase of U content in the Scmgroup first
stem regions was observed. We did not compare U con-
tents of overhangs due to their usually short element
lengths.
Discussion
Here we present a systematic comparison of structural
stability for non-conserved and conserved pre-miRNAs
from human, mouse, and chicken genomes. Previous
studies have compared comparisons between other
kinds of RNAs and native pre-miRNAs, and have pro-
posed that pre-miRNAs are more stable [6,8]. Our
results from comparisons within the pre-miRNA popu-
lation provide novel insights into pre-miRNA thermody-
namic stability and possible links with the pre-miRNA
evolutionary process in animal species. The results pre-
sented here indicated both an upper and lower limit for
pre-miRNA thermodynamic stability, implying a natural
selection pressure against both ultra stable and unstable
pre-miRNAs.
Moderately stable pre-miRNAs in animal could result
from a trade-off between structural rigidity and flexibil-
ity. It is known that secondary structures of pre-miR-
NAs are needed for a correct recognition of specific
enzymes in the miRNA biogenesis [2-5]. Thus maintain-
ing a stable secondary structure could be necessary for
pre-miRNA functioning, which could explain the result
that unstable pre-miRNAs were less favoured by natural
selection. However, the process of miRNA maturing
also involves cleavage and duplex unwinding of pre-
miRNAs [1]. Human Drosha has been reported to selec-
tively cleave pre-miRNA with large terminal loop [18],
which was consistent with our observation that con-
served pre-miRNAs had on average larger terminal
loops than non-conserved pre-miRNAs. It is also known
that duplex unwinding is critical for the processing of
pre-miRNAs to generate mature miRNAs [13,14,16]. As
A-U base pairs were less stable than G-C base pairs, lar-
ger (A-U) % of conserved pre-miRNA could increase
structural flexibility that facilitate the unwinding pro-
cess. Conserved pre-miRNAs also had larger bp % than
non-conserved pre-miRNAs, which could be possibly
ascribed to (1) the influence of bp % on duplex unwind-
ing was minor and/or (2) a trade-off between structure
rigidity and flexibility.
However, the natural selection pressure for pre-
miRNA stability could also involve selection for pre-
miRNA characteristics other than thermodynamic stabi-
lity, but that affect pre-miRNA stability as a side effect.
We have shown that conserved and non-conserved pre-
miRNAs with comparable AMFEs exhibited significant
differences in structural elements. These differences
might be due to a trade-off between pre-miRNA struc-
tural rigidity and flexibility, but the possibility of selec-
tion for factors other than thermodynamics could not
be ignored.
In this study, the enrichment of U nucleotides in con-
served pre-miRNAs was particularly noteworthy. High
pre-miRNA U nucleotide content might both contribute
AUGC
−6%
−4%
−2%
0
2%
4%
6%
Composition difference
Human
AUGC
−4%
−2%
0
2%
4%
Composition difference
Mouse
AUGC
−10%
−5%
0
5%
10%
Composition difference
Chicken
Legend: Sc Sc Sc
1 2 3
A
B
C
Figure 2 Nucleotide composition difference between
conserved and non-conserved pre-miRNAs. The A, U, G, and C
composition difference between conserved (Sc1, Sc2, and Sc3) and
non-conserved pre-miRNAs (Sn) from human (A), mouse (B), and
chicken (C) genomes. The white, gray, and black bars respectively
denote the values that Sc1, Sc2, and Sc3mean nucleotide
compositions minus that of Sn. Sc1for chicken was excluded as it
contained fewer than five pre-miRNAs.
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to maintaining moderate stability and serve as a signal
for miRNA biogenesis [8]. These results provide topics
for future experimental and theoretical investigations,
and raise an interesting theoretical question about the
evolutionary dynamics underlying pre-miRNA structure
and stability. Is the enrichment of U nucleotides in
pre-miRNA the result of step-wise mutation accumula-
tions or filtering from non-conserved pre-miRNAs?
Exploration of this question could provide a deeper
understanding of the miRNA evolutionary process and
underlying mechanism.
As determining the conservation level of pre-miRNA
sequences was critical for our analyses, we chose three
genomes with abundant pre-miRNAs and used dual
conservation constraints to select pre-miRNAs in this
study. Although this method convincingly determined
B E H
C F I
0.4
D G J
0.3
Interior stem
First stem
Terminal loop
Interior loop
overhang
Stem
Loop
A
0
0.2
0.4
0.6
Interior Stem ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.1
0.2
0.3
Interior loop ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.05
0.1
0.15
0.2
0.25
0.3
Overhang ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.2
0.4
0.6
0.8
First stem ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.05
0.1
0.15
0.2
0.25
Terminal loop ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.1
0.2
0.3
0.4
0.5
Interior Stem U ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.2
0.4
0.6
Interior loop U ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.1
0.2
0.3
0.4
0.5
First stem U ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
0
0.2
0.4
0.6
0.8
1
Terminal loop U ratio
Scm (73)
Snu (82)
Snm (109) Sns (69)
Figure 3 Structural element features of conserved and non-conserved pre-miRNAs with different level of stability. (A) diagram of
structural elements of a hairpin secondary structure. (B-F) Boxplot (box with notch) of length ratios of interior stem (B), interior loop (C), terminal
loop (D), first stem (E), and overhang (F) with respect to pre-miRNAs within Scm(black), Snu(red), Snm(red), and Sns(red). (F-J) Boxplot (box without
notch) of region U ratios of interior stem (G), interior loop (H), terminal loop (I), and first stem (J) of pre-miRNAs within Scm, Snu, Snm, and Sns.
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pre-miRNA conservation, it also reduced the size of the
pre-miRNA population used for our investigation. 1,779
sequences of pre-miRNAs were obtained from human,
mouse, and chicken genomes, from which 658 were
selected for further analyses. As more pre-miRNAs are
identified and their sequence conservation determined,
the size of the pre-miRNA population available for
study will increase, allowing for the identification of
stronger general trends in the future. For instance,
investigation of exhaustive miRNA families would allow
us to derive the pre-miRNA evolutionary trajectory by
comparing their thermodynamic stability, nucleotide
compositions, structural features, and mutations from
consensus ancestor sequences.
The results presented here might also be used in the
future to predict or verify pre-miRNA candidates. The
correlation between pre-miRNA thermodynamic stability
and sequence conservation could be helpful for estab-
lishing more comprehensive pre-miRNA filtering criteria
in practical applications. For instance, a loose candidate
sequence filtering constraint could be applied to identify
novel non-conserved pre-miRNAs for a given genome.
On the contrary, a strict constraint for both unstable
and ultra stable secondary structures would reduce the
false positive rate for identifying novel conserved pre-
miRNAs.
Conclusions
In summary, our findings further the understanding of
pre-miRNA thermodynamics, and might facilitate the
investigation on miRNA evolution process. A correlation
was identified between sequence conservation and ther-
modynamic stability for pre-miRNAs from human,
mouse, and chicken genomes. The distribution of
AMFEs for non-conserved pre-miRNAs was significantly
larger than for conserved pre-miRNAs but the overall
mean AMFEs of the two groups were similar. Investiga-
tion of pre-miRNA sequence features was used to
explain their stability distribution. Compared with non-
conserved pre-miRNAs, conserved pre-miRNAs form
more base pairs on average but have a greater propor-
tion of A-U bonds. Furthermore, the variances of
sequence features of conserved pre-miRNAs, such as bp
% and nucleotide composition, were consistently nar-
rower than those for non-conserved pre-miRNAs. Nota-
bly, the U content of conserved pre-miRNAs was higher
than the A, G, or C content, while the non-conserved
pre-miRNAs had more G and C nucleotides, implying
an importance of U nucleotide in pre-miRNA evolution-
ary history.
In addition to thermodynamic stability, we identified
characteristic structural element features of conserved
pre-miRNAs by comparing conserved and non-con-
served pre-miRNAs with comparable stabilities. The
results of this comparison indicated that the natural
selection of pre-miRNA structure and sequence involved
more than thermodynamic stability, indicating that pre-
miRNAs evolutionary is a complex process.
Methods
Data
721, 579, and 479 pre-miRNA sequences of human
(Homo sapiens), mouse (Mus musculus), and chicken
(Gallus gallus) genomes respectively were downloaded
from miRBase (Release 14) [25]. The genome assemblies
for the pre-miRNA coordinates from human, mouse,
and chicken genomes are GRCh37 (Feb 2009, hg19),
NCBIM37 (July 2007, mm9), and WASHUC2 (May
2006, galGal3), respectively.
Pre-miRNA structural stability
The program RNAfold (Vienna RNA package, version
1.7) was utilized with default parameter values to obtain
estimated folding MFEs of pre-miRNAs [26,27]. To
compare the stability of pre-miRNAs with different
nucleotide sequence lengths, we used adjusted MFE
(AMFE) [8]. AMFE is defined as AMFE = -MFE/
(sequence length) × 100. Thus, pre-miRNAs with larger
AMFE values were considered more stable.
Pre-miRNA sequence conservation
The conservation level of pre-miRNA sequences was
determined using two constraints. The first constraint
used was University of California Santa Cruz PhastCons
scores, which measure conservation for each nucleotide
in a specific genome based on a phylogenetic
hidden Markov model in a multiple alignment, for a
given a phylogenetic tree [28,29]. Genomes of human
(GRCh37),mouse(NCBIM37),
(WASHUC2) were aligned against 44, 28, and 5 verte-
brate genomes respectively to generate PhastCons
scores. A pre-miRNA sequence was considered con-
served if the average PhastCons scores in any 15-nucleo-
tide sequence in the hairpin stem region were no
smaller than 0.9 as described by Bentwich et al [19].
A few pre-miRNAs with non-hairpin structure were
disregarded.
To reduce the false positive rate, the conservation of
pre-miRNAs was checked using miRNA family classifi-
cation in miRBase. The miRNA family classifications
were produced by a BLAST-based clustering of all pre-
miRNAs in the database followed by manual curation.
The miRNA family classification provides information
about pre-miRNA homologs. In each species, we filtered
conserved pre-miRNAs, as defined by PhastCons scores,
with few homologs and non-conserved pre-miRNAs
with homologs in unrelated species. We also grouped
the conserved pre-miRNAs into different sets according
andchicken
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to the width of their homolog distribution in the phylo-
genetic tree. M was used to denote the number of taxo-
nomic families where a given miRNA family was
distributed. Conserved pre-miRNAs from miRNA
families with an M < 5 and non-conserved pre-miRNA
from families with an M > 1 were excluded from the
study population. Snwas used to denote the non-con-
served pre-miRNA set containing non-conserved pre-
miRNAs from families with M = 1. As the M values of
miRNA families varied largely, we grouped conserved
pre-miRNAs into three sets, Sci, i = 1, 2, and 3, that
contain conserved pre-miRNAs from miRNA families
with M values of 5 - 9, 10 - 19, and ≥ 20, respectively.
Statistical tests
Pair-wise comparisons were performed for conserved
and non-conserved pre-miRNAs. A two-sample, two-
sided t-test without assumption of equal variance was
used to compare the mean AMFE, nucleotide composi-
tion, and other characteristics. A two-sample two-sided
F-test was applied to compare the distribution variances.
A Lilliefors test [30] was used for testing normality. For
the P-values produced by pair-wise comparison of a
given characteristics between non-conserved and con-
served pre-miRNAs, False Discovery Rate (FDR) control-
ling with Benjamini Hochberg procedure [31] was used
for the multiple-testing correction.
Additional material
Additional file 1: Figure S1. Correlation between pre-miRNA sequence
length and MFE values (A) as well as AMFE values (B). Dashed line is the
linear regression of MFEs with sequence lengths.
Additional file 2: List of pre-miRNA name, length, AMFEs, and
conservation level. This file lists names, lengths, AMFEs, and
conservation level of the pre-miRNAs we studied from human, mouse,
and chicken genomes. The conservation level of 0, 1, 2, or 3 represents
the pre-miRNAs belonging to group Sn, Sc1, Sc2, and Sc3, respectively.
Additional file 3: Table S1. P-values of Lilliefors test for normality [30].
For non-conserved (Sn) and conserved pre-miRNAs (Sc1, Sc2, and Sc3) from
human, mouse, and chicken genomes, Lilliefors test was used to test
normality of AMFE, bp %, (A-U) %, (A+U) %, A%, G%, C%, and U%. The P-
values of test for overall pre-miRNAs from each genome was also given.
* not a normal distribution at the 0.05 level. ** Sc1for chicken was
excluded as it contained fewer than five pre-miRNAs.
Additional file 4: Figure S2. Distribution of bp % and (A-U) % values of
non-conserved (Sn) and conserved pre-miRNAs (Sc1, Sc2, and Sc3) from
human, mouse, and chicken genomes. The number of pre-miRNAs
within each set is indicated in brackets. Sc1for chicken was excluded as
it contained fewer than five pre-miRNAs.
Additional file 5: Figure S3. Distributions of AMFEs of pre-miRNAs
within Scm, Snu, Snm, and Sns.
Acknowledgements
This work was supported by China National 973 programs (2007CB946904),
863 Hi-Tech Research and Development Programs (No. 2007AA02Z311), and
National Nature Science Foundation of China (No. 30700139). We would also
thank the reviewers whose valuable suggestions improved the quality of the
manuscript.
Author details
1Center for Bioinformatics, National Laboratory of Protein Engineering and
Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing
100871, China.2Beijing Institute of Radiation Medicine, Beijing 100850, China.
Authors’ contributions
MN is the major researcher and prepared the manuscript. WS and XB were
involved in data analyses and helped to revise the manuscript. SW and SL
participated in discussion and guided the project. All authors read and
approved the final manuscript.
Received: 29 April 2010 Accepted: 27 October 2010
Published: 27 October 2010
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doi:10.1186/1471-2148-10-329
Cite this article as: Ni et al.: Correlation between sequence conservation
and structural thermodynamics of microRNA precursors from human,
mouse, and chicken genomes. BMC Evolutionary Biology 2010 10:329.
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