Correlation between sequence conservation and structural thermodynamics of microRNA precursors from human, mouse, and chicken genomes
© Ni et al; licensee BioMed Central Ltd. 2010
Received: 29 April 2010
Accepted: 27 October 2010
Published: 27 October 2010
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.
MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate expression at the post-transcriptional level in animals and plants . 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 critical 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 species and one plant species pre-miRNAs have significantly lower estimated folding minimum free energies (MFEs) than those of their shuffled sequences, unlike other kinds of RNAs such as tRNAs, rRNAs , and mRNAs [6, 7]. Zhang et al. directly compared the stability of pre-miRNAs and other kinds of RNAs in seven plant species and showed that pre-miRNAs form more stable secondary structures . 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 . 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 flexibility 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 selectively cleave pre-miRNAs hairpins bearing a large terminal loop (≥ 10 nucleotides) . 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 systematic investigation of pre-miRNA instability has not been carried out. It has been shown that there is a tendency 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 , 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 investigated the correlation between pre-miRNA structural elements and sequence conservation.
Stability comparison between conserved and non-conserved pre-miRNAs
AMFE Comparison between non-conserved and conserved pre-miRNAs from human, mouse, and chicken genomes
S n (132)
44.1 ± 14.1
S c 1 (60)
45.2 ± 9.98
S c 2 (66)
44.6 ± 7.06
S c 3 (33)
44.7 ± 6.36
S n (108)
41.0 ± 9.79
S c 1 (63)
45.1 ± 7.83
S c 2 (75)
45.0 ± 6.93
S c 3 (39)
43.8 ± 5.89
S n (21)
42.2 ± 14.7
S c 2 (40)
41.6 ± 5.59
S c 3 (19)
42.3 ± 5.05
Unlike the mean AMFE values, the AMFE variances of the conserved and non-conserved pre-miRNAs were significantly different in all three species at the FDR 0.05 level (Table 1). Moreover, the AMFE variance consistently decreased from S c 1 to Sc3 in all three species (in chicken S c 1 was excluded). The AMFE standard deviations of S n were 2.21-fold, 1.66-fold, and 2.91-fold larger than S c 3 for 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 S c 3 group, 3.0% (human), 2.6% (mouse), and 0.0% (chicken) of pre-miRNAs were ultra stable. In comparison, in the S n group, 16.7% (human), 9.3% (mouse), and 23.8% (chicken) were ultra stable. Similar results were obtained for unstable pre-miRNAs. In the S c 3 group, 3.0% (human), 2.6% (mouse), and 5.2% (chicken) of pre-miRNAs were unstable, while in the S n group, 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
Summary and comparison of pre-miRNA base pairing and nucleotide composition
S n (132)
68.66 ± 7.83
42.73 ± 16.12
47.11 ± 13.18
22.10 ± 7.89
25.01 ± 6.96
27.49 ± 7.90
25.40 ± 7.24
S c 1 (60)
71.88 ± 6.20A
49.34 ± 11.81Ab
53.87 ± 10.66A
23.78 ± 6.36
30.09 ± 6.42A
24.45 ± 5.53AB
21.68 ± 5.84A
S c 2 (66)
72.94 ± 5.64Ab
48.67 ± 8.17AB
51.62 ± 7.15AB
23.41 ± 4.74B
28.21 ± 4.42AB
25.71 ± 3.49aB
22.67 ± 5.11AB
S c 3 (33)
73.48 ± 4.67AB
50.14 ± 11.02Ab
52.94 ± 9.67a
22.96 ± 6.05
29.98 ± 6.16A
26.06 ± 5.15B
20.99 ± 5.92A
S n (108)
67.87 ± 6.51
44.35 ± 11.20
49.30 ± 9.65
22.40 ± 6.16
26.90 ± 5.97
27.28 ± 5.57
23.42 ± 5.74
S c 1 (63)
72.53 ± 5.85A
48.20 ± 11.47a
53.07 ± 9.42a
23.29 ± 5.42
29.78 ± 6.11A
25.30 ± 5.28a
21.62 ± 5.16a
S c 2 (75)
72.10 ± 5.87A
47.70 ± 8.14aB
50.57 ± 6.77B
22.90 ± 4.57b
27.67 ± 4.40b
26.28 ± 3.72B
23.15 ± 5.02
S c 3 (39)
73.37 ± 5.13A
50.85 ± 10.93A
53.05 ± 9.15a
22.89 ± 5.90
30.16 ± 5.51A
26.06 ± 5.06
20.90 ± 5.53a
S n (21)
68.24 ± 7.37
37.13 ± 17.43
44.17 ± 14.75
19.47 ± 8.79
24.69 ± 8.01
29.35 ± 6.00
26.48 ± 9.74
S c 2 (40)
70.89 ± 7.06
51.22 ± 7.19AB
55.11 ± 4.99AB
24.60 ± 4.14B
30.52 ± 3.97AB
24.80 ± 3.53AB
20.08 ± 3.24aB
S c 3 (19)
73.08 ± 5.14a
53.80 ± 7.56AB
57.18 ± 5.72AB
24.45 ± 5.26
32.74 ± 3.26AB
23.94 ± 4.49A
18.87 ± 3.33AB
Structural element comparison between conserved and non-conserved pre-miRNAs
Although AMFEs for pre-miRNAs in the S c m and S n m 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 increasing sequence stability. Surprisingly, the S c m group exhibited significantly larger interior stem ratios on average than the S n s group (p = 2.5 × 10-5). The S n m group had smaller overhangs than both the S n u and S n s groups, but the overhangs of the S c m group were even significantly smaller than those of the S n m group (p = 3.2 × 10-4). The S c m group also had significantly lower interior loop ratios (p = 3.2 × 10-7) and larger terminal loop ratios than the S n m group (p = 4.4 × 10-2). Only first stem ratios of the S c m and S n m groups 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 S c m group had significantly higher U ratios at interior stem region (p = 3.1 × 10-3) than the S n m group. The S c m group also had higher U content on average than the S n u group at terminal and interior loops, although the differences were not significant. No apparent increase of U content in the S c m group first stem regions was observed. We did not compare U contents of overhangs due to their usually short element lengths.
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 proposed that pre-miRNAs are more stable [6, 8]. Our results from comparisons within the pre-miRNA population provide novel insights into pre-miRNA thermodynamic stability and possible links with the pre-miRNA evolutionary process in animal species. The results presented 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 flexibility. It is known that secondary structures of pre-miRNAs are needed for a correct recognition of specific enzymes in the miRNA biogenesis [2–5]. Thus maintaining 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 . Human Drosha has been reported to selectively cleave pre-miRNA with large terminal loop , which was consistent with our observation that conserved 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, larger (A-U) % of conserved pre-miRNA could increase structural flexibility that facilitate the unwinding process. 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 unwinding 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 stability, 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 structural rigidity and flexibility, but the possibility of selection for factors other than thermodynamics could not be ignored.
In this study, the enrichment of U nucleotides in conserved pre-miRNAs was particularly noteworthy. High pre-miRNA U nucleotide content might both contribute to maintaining moderate stability and serve as a signal for miRNA biogenesis . 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 accumulations 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 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 establishing 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.
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 thermodynamic 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. Investigation 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 proportion of A-U bonds. Furthermore, the variances of sequence features of conserved pre-miRNAs, such as bp % and nucleotide composition, were consistently narrower than those for non-conserved pre-miRNAs. Notably, 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 evolutionary history.
In addition to thermodynamic stability, we identified characteristic structural element features of conserved pre-miRNAs by comparing conserved and non-conserved 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.
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) . 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) . 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), and chicken (WASHUC2) were aligned against 44, 28, and 5 vertebrate genomes respectively to generate PhastCons scores. A pre-miRNA sequence was considered conserved if the average PhastCons scores in any 15-nucleotide sequence in the hairpin stem region were no smaller than 0.9 as described by Bentwich et al . 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 classification 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 to the width of their homolog distribution in the phylogenetic tree. M was used to denote the number of taxonomic 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. S n was used to denote the non-conserved 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, S c i , i = 1, 2, and 3, that contain conserved pre-miRNAs from miRNA families with M values of 5 - 9, 10 - 19, and ≥ 20, respectively.
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 composition, and other characteristics. A two-sample two-sided F-test was applied to compare the distribution variances. A Lilliefors test  was used for testing normality. For the P-values produced by pair-wise comparison of a given characteristics between non-conserved and conserved pre-miRNAs, False Discovery Rate (FDR) controlling with Benjamini Hochberg procedure  was used for the multiple-testing correction.
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.
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