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Abstract
microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 5′ end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families.
Citation: Ibáñez-Ventoso C, Vora M, Driscoll M (2008) Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology. PLoS ONE 3(7): e2818. https://doi.org/10.1371/journal.pone.0002818
Editor: Steven L. Salzberg, University of Maryland, United States of America
Received: January 28, 2008; Accepted: June 19, 2008; Published: July 30, 2008
Copyright: © 2008 Ibáñez-Ventoso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work has been supported by NIH grant R01 AG024882 and R21 AG029376.
Competing interests: The authors have declared that no competing interests exist.
Introduction
microRNAs (miRNAs) are small (16–29 nucleotide (nt)), non-coding RNAs that regulate gene expression at the post-transcriptional level [1]–[5]. Intensive research over the last several years has led to the appreciation that these tiny RNAs act via a highly prevalent, and generally conserved, gene expression regulatory mechanism that impacts development, homeostasis and disease. A major research challenge for the decade will be the elaboration of miRNA function in biology and the investigation of how microRNAs can be exploited for therapeutic application.
To date, little is actually known about the biological functions of most miRNAs—the roles of only a small number have been experimentally elucidated [6], [7]. Numerous studies have reported on miRNA expression profiles in cells, tissues, organisms, and disease states [8]–[31]. In addition, multiple bioinformatic efforts have predicted target mRNA transcripts to suggest candidate genes regulated by miRNA interactions e.g. [13], [32], [33]–[38]. The potential for complex cross-regulation that emerges from these general surveys is staggering, and appreciation for the complexity is further extended by observations that: 1) many mRNA transcripts include potential binding sites for multiple, distinct miRNAs, and 2) different miRNAs that share sequence similarity (especially in the 5′ end seed region) can recognize the same binding sites on individual mRNA targets [39]–[42]. Against this backdrop, the need for understanding when and where miRNAs are expressed, what the relevant mRNA targets are, and what the complete miRNA family sequence relationships encoded by the genome are, is dramatically underscored. This work addresses the latter goal, with an emphasis on invertebrate genetic models that are likely to have a major impact on advancing understanding of miRNA function.
Over the last few years, intensive discovery efforts have contributed to extensive additions and sequence changes to annotated miRBase miRNA compilations for C. elegans, D. melanogaster and humans as total numbers of mature miRNA sequences increased from 107 C. elegans, 79 D. melanogaster and 152 human (miRBase release 3.0, Jan. 2004) to 139 C. elegans, 152 D. melanogaster and 733 human (miRBase release 10.1, December 2007) [43]–[46]. Although it is anticipated that miRNAs will continue to be identified, (numbers of human miRNAs may be in the thousands (see Bentwich et al. [47]), it is likely that most of the abundant miRNAs have been identified in nematodes, flies and humans. Moreover, the majority of identified C. elegans miRNAs have been genetically deleted [48]–[50], an accomplishment that sets the stage for detailed evaluation of functions in this model. Initial studies support that evaluation of functional redundancies will be an important factor in this analysis [39], [40], [42], [48] and that conserved regulatory functions may shed light on disease mechanisms [6], [42]. Thus, we considered it a timely moment to pause and compile an overview of sequence miRNA relationships in invertebrate genetic models.
Given the expanded C. elegans, D. melanogaster and human miRNA identifications and the importance of rapidly identifying potential functional redundancies within and between species, we probed miRNA sequence relationships to compile a current list of mature miRNA sequence families within the C. elegans and D. melanogaster genomes, and we identified their human counterparts. Our analysis presents an overview that significantly expands the memberships of described sequence-related groups within, and between, species. We highlight new sequences conserved between species pairs or among nematodes, flies and humans. This compilation of sequence relationships should facilitate studies on miRNA evolution and conserved function that will contribute to enhanced understanding of complex miRNA regulatory networks and their biological activities.
Results
Recent reports have markedly expanded the numbers of identified miRNAs expressed in C. elegans, D. melanogaster, and humans [10], [11], [25], [47], [51]–[69]. Given the tremendous potential of invertebrate genetics to address in vivo function of conserved miRNAs, the availability of genetic knockouts of most of the 139 reported C. elegans miRNA genes, and our interest in evaluating miRNA contributions to cellular robustness and mechanisms of aging, we sought to generate a current overview of miRNA sequence families identifiable by comparisons among these species. We compared all reported C. elegans 139, D. melanogaster 152 and human 733 mature miRNA sequences available in the miRNA database miRBase 10.1 [43]–[46] using the ClustalW algorithm [70] to identify intra-species and inter-species sequence similarities. We classified miRNAs as sequence-related based on current understanding of functional miRNA-target interactions, which may occur via either of two sequence relationships: 1) perfect complementarity of miRNA 5′ end sequences, especially at nucleotide positions 1,2–8 referred to as the “seed” region, and 2) high level complementarity across the length of the miRNA (>70% identity overall) that can have less precise pairing in the seed region.
5′ end seed region search criteria
5′ end sequences are critical for miRNA function [2], [41], [71]–[73] and the seed region is thought to contribute to target recognition by perfect (or near perfect) complementary binding to the mRNA target site. The requirement for uninterrupted homology may render the miRNA-mRNA seed region under considerable selective pressure. Indeed, seed regions are highly conserved in mRNAs across species [32], [41], [74]. We therefore searched for 5′ end seed matches that featured at least 7 continuous nucleotides of homology within the first 10 nt of the miRNA, a modestly relaxed criteria chosen to provide confidence that most potential seed region sequence relationships would be identified by this search. We did not allow interruptions (mismatches or gaps) within the first 10 nt except base changes that would permit G..U pairing, because G..U base pairing in the seed region has been documented to be possible in vivo under conditions of efficient miRNA target regulation [73].
Homology over miRNA length criteria
Because some miRNAs have less stringent seed region pairing and instead exhibit homology to target transcripts over their lengths [41], [71], we also compiled a list of related miRNAs using the criteria of full-span homology. To establish a reasonable homology cut-off value, we examined previously identified miRNA families and determined that a score of ≥70% sequence similarity over length should identify most, if not all, miRNA homologs known from published target site models and miRNA groups. Because the current mechanism of action of miRNAs has been inferred from only very few validated miRNA-gene targets, we also elected to list miRNAs that exhibit 60–69.9% identity in supporting information. According to current understanding, the potential functional significance of the 60–69.9% similar miRNAs is of higher probability if the homology in the seed region is high.
Overview: Sequence relationships that expand miRNA family lists in nematodes, flies and humans
We performed alignments by both 5′ end seed matching (nt 1–10) and by analysis of homology across complete mature miRNA sequences, comparing individual C. elegans miRNA sequences against all known C. elegans, D. melanogaster and human miRNAs, and individual D. melanogaster miRNAs against D. melanogaster and human miRNAs (Tables 1–6). Our analysis greatly expands the documentation of miRNA sequence family members. For example, our combined list of C. elegans miRNA sequence relationships identified 211 sequence relationships, placing 84 sequence-related miRNAs into 20 family groups (Table 1), whereas previous reports on C. elegans miRNAs [43], [51], [63], [64] identified 110 sequence relationships between 70 miRNAs. Similarly, our combined searches for D. melanogaster miRNAs detected 126 sequence relationships including 70 sequence-related miRNAs in 24 family groups (Table 2), a considerable expansion of the previously reported 53 sequence relationships between 31 miRNAs [25], [43], [52], [57], [60], [65], [75]. Our work increases the number of C. elegans miRNAs with identified human counterparts to 76 (Table 4) and D. melanogaster miRNAs with identified human counterparts to 83 (Table 5) [25], [43], [51], [52], [57], [60], [63]–[65], [75], [76]. Significantly, we associated as many as 133 human miRNA sequences with sequence-related worm and/or fly miRNAs (Tables 4–6 and Figure S1). Below we highlight some details of C. elegans and D. melanogaster miRNA family searches and then discuss the invertebrate miRNAs that have clear human counterparts.
C. elegans miRNA families
C. elegans miRNA families defined by searches for homology in 5′ end sequences.
We searched for 5′ end sequence alignments that included at least 7 nucleotides of continuous similarity within nt 1–10 of the mature miRNA, with no allowed gaps and only G..U mismatches permitted. By these criteria, we identified 81 C. elegans miRNAs that can be placed into 19 different families (Table 1, Dataset S1). We observed that 5′ homologies were mainly located from nucleotides 2 to 8, consistent with conserved sequence present in the seed region (Figure S2). Moreover, related miRNAs sharing longer nucleotide homologies at the 5′ end tend to be more similar at the 3′ end (and therefore over their full lengths) as compared to miRNAs with 5′ homologous regions of only 7 or 8 nucleotides.
C. elegans miRNA families defined by searches for homology over their lengths.
We also compiled a list of miRNA families by requiring homology over the entire miRNA length. We grouped 45 of the 139 C. elegans mature miRNAs into 15 different families based on ≥70% identity over mature sequence length (Table 1, Dataset S2). Consistent with current reports in the field, the highest similarity occurs predominantly at the 5′ end in full-length sequence alignments.
Two homology search criteria generate a C. elegans miRNA family list with substantial, but not complete, overlap.
Combining the two strategies for identification of homologies among miRNAs that we described above, we identify 84 C. elegans sequence-related miRNAs grouped in 20 families (Table 1). This analysis expands the previously reported number of members in C. elegans miRNA families [43], [51], [63], [64] and establishes 1 new sequence-related group containing miRNAs cel-miR-78 and cel-miR-272. About half (101/211) of the sequence relationships described in this work have not been posted in previous works and in the miRBase page listing of sequence relationships among miRNA precursors.
The two homology search approaches we used identify a substantially overlapping list, although clearly not all miRNAs fit both 5′ end and overall similarity criteria. Of the 139 C. elegans miRNAs analyzed, 77 miRNAs exhibit high identity at the 5′ end but <70% overall similarity with at least one of their 5′ sequence-related miRNAs (indicated in Dataset S1). 40 miRNAs have significant homology to sequence-related worm miRNAs only at the 5′ end and thus were not included in the ≥70% homology lists compiled after full length sequence comparison (Table 1, Dataset S2). Conversely, not all miRNAs with similarity over the sequence length include 7 or more continuous identical nucleotides within the first 10 nt of the 5′ end. 3 of the 45 miRNAs with ≥70% identity (cel-miR-78, cel-miR-270 and cel-miR-272) fail to comply with our criteria for 5′ end family grouping and therefore are not included in the list of 5′ end-related miRNAs in Dataset S1.
3′ end sequences
miRNA target sites with perfect complementarity to miRNA 3′ ends and negligible pairing at the 5′ end have not been described—extensive 3′ pairing has been suggested to act as a determinant of target specificity or regulatory sensitivity within miRNA families [41], but it is the 5′ end sequences that are thought to drive target selection and major regulation. Nonetheless, we were curious as to whether miRNAs could share significant sequence similarity at the 3′ end but negligible or weak 5′ similarity. We therefore probed relationships among 3′ end sequences of mature C. elegans miRNAs by multiple alignments of the 3′ sequence of each miRNA against 3′ sequences of all the remaining miRNAs. About half of C. elegans miRNAs are ≥60% similar to another at their 3′ end (67/139); one quarter of these are >70% identical. In general, however, the more nucleotide similarity at the 3′ end, the more identical the miRNAs are at the 5′ end.
It may be noteworthy that within the group of miRNAs with 50–70% 3′ similarity, we could identify some with extensive sequence identity at the 3′ end and low 5′ similarity (Figure S3). These groups are: 1) cel-lin-4, cel-miR-87; 2) cel-miR-90, cel-miR-124 (3′ region of identity also conserved to some extent in cel-miR-80, cel-miR-81, cel-miR-82 and cel-miR-234); 3) cel-miR-81, cel-miR-799 (3′ region of identity also conserved to some extent in cel-miR-80 and cel-miR-82); and 4) cel-miR-52, cel-mir-53, cel-miR-70, cel-miR-229 and cel-miR-272. Although no data are yet available to address the potential functions of these 3′-related miRNAs, their conservation suggests these 3′ motifs could be important for miRNA function. For example, a hexanucleotide 3′ terminal motif has recently been shown to direct hsa-miR-29b to the nucleus [77].
Searches of the C. elegans 3′ miRNA motifs in Drosophila and humans identified 3′ relationships of cel-miR-80 and cel-miR-799 with hsa-miR-208a, and interestingly revealed 3′ relationships of hsa-miR-208a with hsa-miR-129-3p and hsa-miR-129* and of hsa-miR-124 with hsa-miR-377* (Figure S3). Thus, 3′ homologous sequences might reveal functional similarities among miRNAs in nematodes, flies and humans.
Overall, although some 3′ end similarities can be distinguished among miRNAs (even for miRNAs placed into different families), our overview of 3′ end homologies among miRNAs strongly supports the current idea that 5′ end miRNA sequences are much more highly conserved than 3′ ends.
Clustering of mir genes in C. elegans and D. melanogaster genomes
miRNAs can derive from their own transcription units or from exons or introns of other genes [78]. Consecutive mir genes with the same transcriptional orientation within relatively short distances can be considered as clustered. 42% of human mir genes appear in clusters of 2 or more within 3 Kb intervals [79].
Some C. elegans mir gene clusters have been previously described: mir-35-mir-41 (within a 796 bp region), mir-42-mir-44 (307 bp), mir-54-mir-56 (403 bp), mir-229_mir-64-mir-66 (754 bp), mir-73-mir-74 (374 bp), and mir-241_mir-48 (∼1.7 Kb) [61], [63], [80]. Genes within these groups exhibit similar expression patterns, indicating that they might be co-transcribed into polycistronic units. Based on these observations, we chose a potential clustering range of 2 Kb to evaluate relative mir gene distribution in the C. elegans genome (Figure 1A). Interestingly, 35/137 C. elegans mir genes cluster into a total of 13 groups by this criterion. Most of the clusters contain 2 mir genes, with clustered mir genes more abundant on chromosomes II and X (the latter of which harbors a higher than average number of mirs overall (Figure S4A)). We checked whether clustered mirs are related in sequence and found that about half of the mir clusters contain mir genes that are homologous at the 5′ end and/or over full length (≥70%). If co-expressed, such genes might regulate common mRNAs by recognizing the same target sites. mirs in the remaining half of the clusters do not exhibit significant homology between them. If these mirs are co-expressed, they may target different mRNAs or might interact with the same target transcripts via multiple, distinct miRNA binding sites.
35 of the 137 C. elegans mir genes (the 137 genes produce 139 miRNA forms) (A) and 60 of the 152 D. melanogaster mir genes (B) are situated within 2 Kb of each other on one of their chromosomes (6 chromosomes in C. elegans: Chr. I–V, Chr. X; 4 pairs of chromosomes in D. melanogaster: Chr. 2–4, X/Y). ∼63% clustered mir genes in the C. elegans genome and ∼38% in the D. melanogaster genome are related in sequence. Bounding boxes highlight clustered mir genes of conserved sequences at the 5′ end (ˆ) and/or over full length (#70 indicates ≥70% similarity) (see Tables 1, 2 and Datasets S1, S2, S4 and S5 for details). mir genes on the left or above chromosomes are found in the Watson strand whereas those on the right or below are located in the Crick strand. The physical centers of C. elegans chromosomes are indicated by “0”.
We also looked at the distribution of mir genes in the D. melanogaster genome (Figure 1B). Consistent with previous reports [52], [60], we determined that 60/152 Drosophila mir genes are clustered into 20 different regions 2 kb long. Clusters contain on average 3 mir genes with the longest cluster including 8 mir genes. Clustered mir genes are more abundant on chromosomes 2L and 2R, which also have a higher than average number of mirs overall (Figure S4B). ∼38% of the clustered mir genes in the Drosophila genome have 5′ and/or ≥70% full homologous sequences.
D. melanogaster miRNA families
Similar to our strategy for C. elegans miRNA analysis, we screened D. melanogaster miRNAs for 7 consecutive identical nucleotides at the 5′ ends and classified 61 miRNAs into 19 families (Table 2, Dataset S4). Using the criteria of ≥70% overall identity, we highlight a total of 38 miRNAs that can be classified into 14 families (Table 2, Dataset S5). Overall, we identified 70 of the 152 Drosophila miRNAs as part of 24 sequence-related groups (Table 2).
As is the case for C. elegans miRNAs, lists of related Drosophila miRNAs compiled by the 5′ and ≥70% search criteria overlapped. However, 48 fly miRNAs are significantly similar at their 5′ end but have <70% overall identity with at least one of their sequence-related miRNAs (indicated in Dataset S4). Of these, 33 miRNAs have significant homology to other fly miRNAs only at their 5′ end and thus are not listed in the ≥70% homology groups (Table 2, Dataset S5). Most of the fly ≥70% full length homologs exhibit blocks of ≥7 nt identity at the 5′ end except the following 10: dme-miR-10, dme-miR-100, dme-miR-263a, dme-miR-263b, dme-miR-954, dme-miR-966, dme-miR-1009, dme-miR-1010, dme-miR-iab-4-3p and dme-miR-iab4as-3p.
miRNAs conserved between C. elegans and D. melanogaster
We next compiled an expanded list of sequence-related miRNAs common to nematodes and flies. We searched for both 5′ end matches and for ≥70% homology over extended length between the 139 C. elegans and 152 D. melanogaster miRNAs using the criteria we described above for intra-species comparison. Overall, our sequence comparisons establish 64 novel worm/fly miRNA relationships [25], [43], [51], [52], [57], [60], [63]–[65], [75] and identify 87 miRNA families that now include 87 C. elegans and 65 D. melanogaster members (Table 3).
5′ end homology searches detected 87 worm miRNAs related to 62 fly miRNAs (Table 3, Dataset S7), whereas ≥70% overall identity searches highlighted 31 worm miRNAs and 37 fly miRNAs in family relationships (Table 3, Dataset S8). Of the 87 5′ related C. elegans miRNAs, 68 have a ≥7 nt block homology at the 5′ end but weak full length identity (<70%) with at least one of their 5′ fly miRNA relatives (indicated in Dataset S7). 59 of these have <70% full length sequence similarity with all their 5′ Drosophila relatives and thus these relationships are not present in our ≥70% homology lists in Dataset S8. 15 of the 87 C. elegans miRNAs with 5′ identities in flies have significant extended homology over their full length (≥70%) with all their Drosophila counterparts. Most of the C. elegans_Drosophila ≥70% miRNA homologs have ≥7 nt identity at the 5′ end except cel-miR-239a_dme-miR-12, cel-miR-252_dme-miR-252 and cel-miR-250_dme-miR-1007.
miRNAs conserved between C. elegans and H. sapiens
We also searched for both 5′ end identities and for homologous (≥70%) extended sequence between C. elegans (139) and human (733) miRNAs using the criteria we described above. Overall, our sequence comparisons establish 141 novel nematode_human relationships [43], [51], [63], [64], [76] and identify 76 miRNA families that now include 76 C. elegans and 102 human members (Table 4). 76 worm miRNAs exhibit significant homologies to the 5′ ends of 98 human miRNAs (Table 4, Dataset S10), whereas 22 nematode miRNAs are ≥70% homologous over their full length to 46 human miRNAs (Table 4, Dataset S11). 69 of the 76 5′ related C. elegans miRNAs have <70% extended homology with at least one of their 5′ human counterparts (shown in Dataset S10), and 54 are weakly similar (<70%) with human miRNAs outside their 5′ end sequences. 7 of the above 76 C. elegans miRNAs have significant 5′ and overall (≥70%) homology with all their 5′ related sequences in humans. In our set of C. elegans_human ≥70% homologs, the following do not have ≥7 nucleotides of continuous similarity at the 5′ end: cel-miR-57 with hsa-miR-99a and hsa-miR-100; cel-miR-236 with hsa-miR-141 and hsa-miR-200a; and cel-miR-266 with hsa-miR-25*, hsa-miR-301a and hsa-miR-301b.
miRNAs conserved between D. melanogaster and H. sapiens
Looking for 5′ end and ≥70% overall sequence similarities between D. melanogaster (152) and human (733) miRNAs, we detected 149 novel sequence relationships previous reported in [25], [43], [52], [57], [60], [65], [75], [76] expanding family groups to 83 defined by 83 Drosophila miRNAs and 121 human miRNAs (Table 5). Specifically, 82 Drosophila miRNAs show significant 5′ sequence identity to 117 human miRNAs (Table 5, Dataset S13), and 40 fly miRNAs are ≥70% homologous over full length to 56 human miRNAs (Table 5, Dataset S14). 67 of the above 82 Drosophila miRNAs are <70% identical to the full sequences of some of their 5′-related human miRNAs (identified in Dataset S13)—45 are weakly similar (<70%) to all their 5′ related human sequences outside the 5′ region. The remaining 15 of the 82 Drosophila miRNAs have ≥70% overall homology in addition to 5′ relation to all their 5′ human counterparts. 8 of the 40 Drosophila miRNAs with ≥70% homologous sequences in humans show extensive overall similarity with 5′ mismatches: dme-miR-8 with hsa-miR-141 and hsa-miR-200a, dme-miR-10 with hsa-miR-100 and hsa-miR-99a, dme-miR-100 with hsa-miR-10a and hsa-miR-10b, dme-miR-125 with hsa-miR-10a and hsa-miR-10b, dme-miR263a with hsa-miR-183, dme-miR-263b with hsa-miR-183, dme-miR-306 with hsa-miR-873, and dme-miR-993 with hsa-miR-100*.
miRNAs conserved among nematodes, flies and humans
miRNAs that are conserved among nematodes, flies and humans are likely to regulate biological functions common between invertebrates and vertebrates. Thus, we had considerable interest in identifying miRNAs that are conserved in these three organisms. We found a total of 73 C. elegans miRNAs with identifiable sequence related counterparts shared by nematodes, flies and humans, summarized in Table 6, Venn diagram of Figure 2 and Figure S1.
73/139 C. elegans miRNAs share 5′ end identities and/or ≥70% homology over sequence with miRNAs both in fly and humans. 13 C. elegans miRNAs currently appear to have sequence-related miRNAs limited to C. elegans, 14 miRNAs are shared by nematodes and flies, and 3 miRNAs are shared by nematodes and humans. For Drosophila, 54/152 miRNAs have 5′ and/or ≥70% overall homology to nematode and human miRNAs. 15 D. melanogaster miRNAs have sequence-related sequences restricted to fly, 11 miRNAs appear present both in fly and nematodes and 29 in fly and humans. Names of family members cross species can be found in Tables 1–6 and sequence alignments in supporting datasets and Figure S1. Human miRNAs that have family members only in human are not included. It should be noted that the Venn diagram is inclusive showing miRNAs that have 5′ and ≥70% overall conserved sequences as well as miRNAs with either 5′ or ≥70% overall conserved sequences. Thus, miRNA totals in the diagram sections do not necessarily match those stated in the main text referring only to 5′ sequence identity or only to ≥70% overall homology. Moreover, dme-miR-3, dme-miR-12 and dme-miR-318 are listed in both fly_nematode and fly_human groups but not in the fly_nematode_human group because their corresponding C. elegans and H. sapiens homologs are not cross related in sequence under our criteria. Similarly, dme-miR-3, dme-miR-12, dme-miR-263a and dme-miR-318 are included in both fly_nematode and fly_human groups but not in the fly_nematode_human group when only 5′ homology is considered (main text).
Limiting our relationship criteria to 5′ end sequence identities, we identified 73 C. elegans miRNAs with 5′ homologs in both flies and humans (Table 6, Figure S1). Some C. elegans miRNAs have conserved 5′ ends either in flies or humans: 14 nematode miRNAs have 5′ homologs in flies and 3 have 5′ homologs in humans. 10 nematode miRNAs have similar 5′ ends with other C. elegans miRNAs that have not yet been found among fly or human miRNAs.
Using extended homology search criteria, we identified 16 C. elegans miRNAs that exhibit ≥70% sequence identity with both fly and human counterparts (Table 6, Figure S1). We found that 15 C. elegans miRNAs have ≥70% homologous counterparts in flies that are not found in the human genome, possibly lost during evolution of complex higher organisms, or possibly remaining to be discovered in human genomes. 6 nematode miRNAs have ≥70% homologous counterparts in humans but currently lack identifiable family members in Drosophila. 28 C. elegans miRNAs have ≥70% sequence similarity with other C. elegans miRNAs but were not found in either fly or human genomes.
In a similar manner, we inspected the conservation of D. melanogaster miRNAs in nematodes, flies and humans. 54 D. melanogaster miRNAs have homologous sequences both in nematodes and humans (Figure 2). Searches with 5′ end sequences identified 54 D. melanogaster miRNAs with 5′-related sequences in both nematodes and humans, 9 in nematodes and 29 in humans. 11 D. melanogaster miRNAs have 5′ related sequences only in flies and are not present or remain unidentified in nematodes and humans. Considering ≥70% identity over the entire length, 21 D. melanogaster miRNAs have ≥70% homology counterparts in both nematodes and humans, 16 in nematodes and 19 in humans. 15 D. melanogaster miRNAs have ≥70% similarity to only other fly miRNAs.
Overall, analysis of most recent miRBase release data highlights significant conservation of many miRNAs, supporting that analysis of their biological activities in invertebrate models will shed insight into functions relevant to human biology.
Discussion
An overview of inter- and intra-species relationships among miRNAs
miRBase release 10.1 (December 2007) identifies 733 human, 139 C. elegans and 152 D. melanogaster mature miRNAs [43]–[46]. This list of annotated miRNAs, compiled predominantly from large-scale sequencing studies, has grown at an impressive rate in the recent past–for example, the list of human miRNAs has increased by over 500 sequences during the last 3 years. Although miRNA identification efforts are unlikely to yet be complete, current documented miRNAs most likely represent abundant species processed from typical hairpin structures. The field now faces the challenge of determining the biological activities of these miRNAs. Recently, extensive collections of C. elegans mir mutants have been generated [48], defining an opportune moment at which to evaluate sequence-related families and conserved functions.
In this paper, we present a comprehensive classification of all the miRBase 10.1 miRNA sequences annotated in C. elegans, D. melanogaster and humans into sequence-related groups to identify miRNAs with possible redundant functions in the same species and those with potentially conserved functions across species. This compilation, which takes into account the two ways in which functionally related mature miRNAs can be similar (either 5′ end seed homology or homology over length), is based on mature miRNA sequences rather than precursor gene sequence and adds to the considerable numbers of documented sequence-related family members [25], [43], [51], [52], [57], [60], [63]–[65], [75], [76], providing details of sequence relationships.
Intraspecies analysis: many invertebrate miRNAs have potential for functional redundancy
Looking within individual species, we find that ∼60% (84/139) C. elegans miRNAs and ∼46% (70/152) D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes. The potential for functional redundancy of miRNAs is clearly considerable within these species.
The importance of evaluating sequence-related miRNAs during functional analysis has been elegantly exemplified by work on the C. elegans let-7 miRNA family. Sequence-related miR-48, miR-84 and miR-241 work together to regulate developmental timing by redundant complementarity to binding sites in the 3′ UTR of hbl-1 [40]. mir-48, mir-84 and mir-241 single mutants are seemingly wild type at 20°C. However, double and triple combinations of mir-48, mir-84 and mir-241 mutations cause developmental defects, revealing biological roles for these family members and stressing the importance of the analysis of multiple homologous miRNAs during functional studies. Of course, sequence-related miRNAs might be expressed in different tissues or at different times in development, and therefore might be excluded from performing similar functions with common targets. Still, the extensive sequence relationships that we document underscore that potential co-expression of sequence-related miRNAs will be a significant factor in evaluation of genetic disruptions as well as in commonly executed over-expression studies. Information on the expression patterns of sequence-related miRNAs will be important to careful interpretation of experimental outcomes.
The extent of conservation of miRNA sequences from invertebrates to humans is striking
Another theme that our analysis underscores is the substantial conservation of miRNA sequences across species. ∼62% C. elegans miRNAs are related to Drosophila miRNAs (87/139), ∼55% C. elegans miRNAs are related to human miRNAs (76/139), and ∼55% Drosophila miRNAs are related to human miRNAs (83/152). Over half of the C. elegans miRNAs share sequence homology with miRNAs expressed in both flies and humans (73/139), and this number should increase with an increase in reported miRNAs.
The extensive conservation across species suggests that this group of miRNAs contributes important functions in biology and that experiments in one species may well inform on the biology of another. Indeed, cross-species analyses of let-7 miRNA function has already provided useful leads for addressing human disease regulation. let-7 represses C. elegans RAS ortholog let-60 [81], as well as the human RAS oncogene transcript [42]. Recently these findings have been extended to demonstrate that let-7 expression reduces tumor growth in mouse lung tumor models [82].
Taking stock in a dynamic field
miRNA discovery is an highly active research area. Here we report 133 human miRNAs with related sequences encoded by the C. elegans and/or D. melanogaster genomes. The majority of cataloged human miRNAs have unknown functions. Gene knock-outs, chemically modified antisense oligonucleotides, decoy miRNA targets (miRNA sponges) and over-expression studies are currently being used to evaluate loss-of-function of miRNAs [48], [83]–[86]. Functional investigation of sequence-related miRNAs from C. elegans and D. melanogaster in a whole-organism context will most certainly provide insight into miRNA roles in specific mechanisms relevant to normal development as well as disease. The numerous sequence relationships identified to date will help focus research on abundantly expressed, conserved miRNAs while additional miRNA discovery continues to expand known miRNA families.
Methods
miRNA sequences and criteria for family grouping
Mature miRNA sequences in C. elegans, D. melanogaster and H. sapiens were retrieved from the miRNA registry release 10.1 (December 2007) in miRBase [43]–[46]. miRNAs in C. elegans, D. melanogaster, C. elegans-D. melanogaster, C. elegans-H. sapiens and D. melanogaster-H. sapiens were classified into homology groups based on their sequence similarity at the 5′ end (nucleotides 1–10) and/or over full length. 5′ end sequences (10 nt) were considered homologous when they exhibited identity over 7 continuous nucleotides. Only interruptions implying G..U pairing were allowed within the 7 nt identity block. ≥70% overall similarity was the threshold used for grouping full miRNA sequences into families. miRNAs were thus classified as members of a specific family group if they met the criteria of 5′ 7 nt identity or ≥70% overall similarity with a at least one other miRNA member of the group. The sub-groups noted in supporting information contain miRNAs with more closely similar sequences (≥80% overall identity or highly similar 5′ ends). Expanded groupings of miRNAs exhibiting 60–69.9% sequence similarity are also included in supporting information to provide access to potentially related sequences that might be relevant to a given study. 3′ similarity searches were performed with 3′ end sequences (nucleotides 11-3′ end) of C. elegans miRNAs.
miRNA sequence analysis
Analysis of mature miRNA sequences was performed using Clustal X 1.83 [87] and AlignX (a component of Vector NTi Advance 10.3.0, Invitrogen), which are both based on the Clustal W algorithm [70]. Intraspecies sequence-related miRNAs in C. elegans and D. melanogaster were evaluated by manual examination of multiple sequence alignments and 1000 bootstrapped NJ-trees. Interspecies sequence-related miRNAs were identified by manual inspection of profile alignments, in which all D. melanogaster or H. sapiens miRNA sequences were aligned against each of the 139 C. elegans miRNAs (used as reference sequence) in C. elegans-D. melanogaster and C. elegans-H. sapiens analyses, and all H. sapiens miRNA sequences were aligned against each of the 152 D. melanogaster miRNAs (reference) in the D. melanogaster-H. sapiens analysis.
mir gene clusters
Coordinates of mir genes in the C. elegans and D. melanogaster genomes were obtained from miRBase release 10.1, December 2007 [43]–[46]. mir genes were considered to form part of a cluster if they were positioned on the same DNA strand within a 2 Kb region. Diagrams were designed using Vector NTi Advance 10.3.0 (Invitrogen).
Supporting Information
Figure S1.
Alignments of miRNA sequences conserved across species. See Table 6 and Figure 2. Grey shading identifies potential G..U pairing.
https://doi.org/10.1371/journal.pone.0002818.s001
(0.06 MB PDF)
Figure S2.
Frequency and distribution of 5′ homologous nucleotides and their correlation with overall sequence conservation. A: Analysis of all 5′ sequence-related miRNAs in C. elegans indicates that homologous nucleotides are mainly positioned from nucleotides 2 to 8 (Dataset S1). B: Sequence-related miRNAs with 7 or 8 homologous nucleotides at the 5′ end tend to have poorer sequence similarity at the 3′ end and thus weaker overall similarity than related miRNAs with 9 or 10 5′ nucleotide homologies. miRNAs with 10 5′ homologous nucleotides tend to have significant nucleotide similarities at the 3′ end with an overall sequence identity of 70–100% (Table 1, Dataset S2) or less frequently of 60–69.9% (Dataset S3).
https://doi.org/10.1371/journal.pone.0002818.s002
(0.32 MB PDF)
Figure S3.
Sequence alignments of C. elegans miRNAs with extensive similarity at the 3′ end but poor homology at the 5′ end. C. elegans mature miRNAs vary in length from 18 nt to 26 nt, and thus the 3′ end sequences used differed in length to some extent. Since the majority of C. elegans miRNAs are 21–23 nt long and on average 22 nt, most of the 3′ end sequences varied 1–2 nt in size. Grey shading denotes potential G..U pairing.
https://doi.org/10.1371/journal.pone.0002818.s003
(0.02 MB PDF)
Figure S4.
Average distribution of mir genes in the C. elegans and D. melanogaster genomes. Despite additional mir genes might still be discovered further concentrating genetic maps, it is worthy of note that a higher proportion of mir genes in miRBase 10.1 are located in C. elegans chromosome X (A) and in Drosophila chromosome pair 2L, 2R (B) than expected by random distribution. The expected number of mir genes was determined by dividing total number of mirs in the genome by chromosome length.
https://doi.org/10.1371/journal.pone.0002818.s004
(0.39 MB PDF)
Dataset S1.
Homology table and sequence alignments of C. elegans miRNAs with significant identity at the 5′ 10 nt.
https://doi.org/10.1371/journal.pone.0002818.s005
(0.39 MB DOC)
Dataset S2.
Homology table and sequence alignments of C. elegans miRNAs with ≥70% overall sequence identity.
https://doi.org/10.1371/journal.pone.0002818.s006
(0.25 MB DOC)
Dataset S3.
Table and alignments of related C. elegans miRNAs with 60–69.9% overall sequence similarity.
https://doi.org/10.1371/journal.pone.0002818.s007
(0.15 MB DOC)
Dataset S4.
Homology table and sequence alignments of D. melanogaster miRNAs with similar 5′ ends.
https://doi.org/10.1371/journal.pone.0002818.s008
(0.12 MB DOC)
Dataset S5.
Homology table and alignments of D. melanogaster miRNAs showing ≥70% overall sequence identity.
https://doi.org/10.1371/journal.pone.0002818.s009
(0.08 MB DOC)
Dataset S6.
Table and alignments of D. melanogaster miRNA sequences with 60–69.9% similarity.
https://doi.org/10.1371/journal.pone.0002818.s010
(0.15 MB DOC)
Dataset S7.
Homology table and alignments of C. elegans miRNAs related at the 5′ end to Drosophila miRNAs.
https://doi.org/10.1371/journal.pone.0002818.s011
(0.32 MB DOC)
Dataset S8.
Identity table and alignments of C. elegans miRNAs with ≥70% full sequence homology to Drosophila miRNAs.
https://doi.org/10.1371/journal.pone.0002818.s012
(0.09 MB DOC)
Dataset S9.
Table and alignments of C. elegans and Drosophila miRNAs with 60–69.9% similarity over whole sequence.
https://doi.org/10.1371/journal.pone.0002818.s013
(0.21 MB DOC)
Dataset S10.
Tables and alignments of C. elegans and H. sapiens miRNAs with homologous 5′ ends.
https://doi.org/10.1371/journal.pone.0002818.s014
(0.47 MB DOC)
Dataset S11.
Sequence identity table and alignments of C. elegans-H. sapiens miRNAs with ≥70% overall homology.
https://doi.org/10.1371/journal.pone.0002818.s015
(0.12 MB DOC)
Dataset S12.
Similarity table and sequence alignments of C. elegans-H. sapiens miRNAs with 60–69.9% overall identity.
https://doi.org/10.1371/journal.pone.0002818.s016
(0.24 MB DOC)
Dataset S13.
Table and alignments of D. melanogaster and human miRNAs with 5′ homology.
https://doi.org/10.1371/journal.pone.0002818.s017
(0.44 MB DOC)
Dataset S14.
Table and alignments of D. melanogaster and human miRNAs with ≥70% overall sequence homology.
https://doi.org/10.1371/journal.pone.0002818.s018
(0.15 MB DOC)
Dataset S15.
Similarity table and sequence alignments of D. melanogaster-H. sapiens miRNAs with 60–69.9% overall identity.
https://doi.org/10.1371/journal.pone.0002818.s019
(0.31 MB DOC)
Author Contributions
Conceived and designed the experiments: CIV MV MD. Performed the experiments: CIV MV. Analyzed the data: CIV MV. Wrote the paper: CIV MD.
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