miRNA Profile of a Triassic Common Mammalian Ancestor and Pre- miRNA Evolution in the Three Mammalian Reproductive Lineages

MicroRNAs (miRNAs) are major factors in the regulation of gene expression. Recent evolutionary studies of miRNAs indicate that important biological innovations, such as the advent of bilateral symmetry and placental reproduction, are accompanied by bursts of miRNA creation which are subsequently conserved via purifying selection. The emergence of eutherian (placental) mammals followed by as much as fifty million years the appearance of the first true mammals in the late Triassic, some 230 million years ago. We have utilized microRNA inventories of eutherian, metatherian (marsupial), monotreme (platypus), and chicken genomes to assemble a minimal microRNA profile of the last common ancestor of all mammals consisting of 171 miRNAs. This profile suggests that the rise of placental reproduction launched a more than three-fold expansion of microRNAs. In addition to expansion of the microRNA repertoire, the conserved microRNAs from five mammalian and one avian genome show evidence for conforming to a canonical phylogenetic history as well as dramatic deviations from the assumptions of molecular clock-like rates and the equality of substitution rates among lineages. We also show that many of these basal mammalian miRNAs are highly expressed in eutherian placenta thus creating an opportunity to gain insight on how microRNAs acquire new targets and new functions. INTRODUCTION Molecular paleontology, or molecular phylogeny, is the use of comparative genomics on extant species coupled with knowledge of the fossil record to infer molecular characteristics of their extinct ancestors as well as to detail evolutionary events that have occurred between extant species and their last common ancestors. If both sources of information are reliable, there are useful, important, and even unique insights to be had. Nowhere has this been more true than in evolutionary developmental biology, or evo devo for short, in which organism-level development and the genetic factors responsible for it are being traced over hundreds of millions of years [1, 2]. One aspect of development that has emerged over the last few years to assume a crucial position is the role of small, non-coding RNAs, in particular the microRNAs (miRNAs), and this has not escaped the notice of those equipped to venture into the realm of molecular paleontology. Sempere et al. [3] presented an evolutionary history of 292 non-paralogous Metazoan microRNAs (miRNAs). They observe a large number of microRNAs arising among certain taxa such as bony fishes and eutherian (placental) mammals which is consistent with other observations that major developmental events, like the advent of bilateral symmetry, the emergence of the vertebrates, and the placental reproduction strategy among mammals, were each accompanied by expansions in the microRNA repertoire of the relevant taxa [4-6]. However, none of these studies was able to take advantage of the non-eutherian mammalian genomes that have recently become available. *Address correspondence to this author at the Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa 52241, USA; Tel: 319-6268450; E-mail: [email protected] We recently reported on an in silico and in vitro miRNA survey of the marsupial species Monodelphis domestica [7]. Here we have updated that survey and have used the updated data to carry out an in silico miRNA survey of another marsupial genome, the tammar wallaby (Macropus eugenii), and the genome of the platypus (Ornithorhynchus anatinus), one of only two monotreme species still extant. These miRNA data, coupled with the most recent miRNA database releases for the human (Homo sapiens), mouse (Mus musculus), and chicken (Gallus gallus) genomes [8], provide us with the opportunity to employ the principles of comparative genomics to simultaneously address two aspects of miRNA evolution. The first of these is an initial attempt to create what could be called a minimal miRNA profile of the last common ancestor of the Mammalia that the paleontologic record suggests lived in the Upper Triassic, some 230 million years ago. This profile consists of 171 miRNAs that are shared among placental, marsupial, and monotreme genomes. The second aspect we have addressed, based upon a subset of the comparative data that includes the chicken miRNAome, is a detailed look at phylogenetic patterns and evolutionary rates not only for the complete miRNA precursor sequences shared among the six species, but also for partitions of those precursors that we have designated by their function within the pre-miRNA hairpin. Results of these analyses indicate that, while overall canonical phylogenetic relationships among the six species are recovered using complete premiRNA sequences, partitioned pre-miRNA sequences that comprise non-expressed fold back regions opposite mature miRNAs and hairpin sequences that are neither expressed nor in the loop, deviate slightly from canonical. We also find that substitution rates vary considerably between species within hairpin partitions and that only the fold back region displays molecular clock-like behavior. miRNA Profile of a Triassic Common Mammalian Ancestor The Open Genomics Journal, 2008, Volume 1 23 MATERIALS AND METHODS An Updated Monodelphis domestica miRNA Screen The in silico strategy followed in the original M. domestica miRNA screen [7] was also followed here using the most current M. domestica genome assembly (MonDom5) and release 10.0 of miRBase. Briefly, pre-miRNA sequences for chicken, mouse, and human were screened in MonDom5 using BLAST. The two criteria used for accepting a hit in the M. domestica genome were a minimum 90% length match and a minimum 90% sequence identity over the pre-miRNA sequence. All accepted miRNA matches were then validated by a search of miRBase with the M. domestica pre-miRNA sequence to establish identity and a comparison with the miRNA map location in the human genome via M. domestica/H. sapiens syntenic maps compiled at the Broad Institute (M. Kamal, personal communication) to establish synteny. We also validated the mature miRNA sequences using a 95% sequence match minimum. M. domestica-Based M. Eugenii and O. anatinus miRNA Screens Once the updated M. domestica pre-miRNA file was completed and validated, the entire pre-miRNA sequence file was screened against the current whole genome sequences of the tammar wallaby (M. eugenii) in GenBank Trace Archive and the platypus (O. anatinus) genome assembly in Ensembl (Oana-5.0). The same criteria used for accepting matches in the opossum genome were applied to the wallaby and platypus genomes when using the opossum pre-miRNA sequences as queries. Validation consisted of a search of candidate pre-miRNAs in miRBase only for these species since the current state of the two genomes, particularly the wallaby genome, does not permit employing the syntenic chromosome mapping criterion as was done for M. domestica. Sequence Alignments, Partition Assignment, Phylogenetic Hypotheses, Rate Tests We were able to assemble a subset of 94 pre-miRNAs that are shared among the genomes of chicken, platypus, wallaby, opossum, mouse, and human. These pre-microRNA sequences were aligned among genomes using Clustal W [9]. Once aligned, we partitioned the pre-miRNAs based on a scheme that reflects the function of the subsequences within the hairpin structure. Partition 1 consists of only mature miRNA sequences. Partition 2 consists only of that part of the hairpin that is clearly a loop sequence as shown in miRBase for the human miRNA. Partition 3 consists of the fold back part of the hairpin directly opposite the mature miRNA sequence. Finally, Partition 4 consists of any remaining sequence in the hairpin. We made a distinction based, again, on the human miRNA in miRBase between a fold back (Partition 3) and a “star” sequence. Since star sequences lie opposite the mature miRNA and are expressed, at least as far as has been reported in mouse and human miRNAs, and appear to play a role in both miRNA and target-site evolution [10], we placed them in Partition 1. Again, for clarity, we reserve the term “fold back” for Partition 3 sequences to distinguish them from the expressed Partition 1 “star” sequences. Phylogenetic reconstructions and hypotheses were constructed and tested using the PHYLIP package [11], specifically the programs dnaml and dnamlk. Rates among sites were treated as gamma distributed with an alpha = 0.25 and 4 rate categories, where these parameters were determined empirically from the present data. There are 945 binary rooted trees for 6 taxa and maximum likelihood topologies resulted from 10-fold jumbling the input order of the 6 taxa with global rearrangements and optimization. Bootstrap values are the result from 100 bootstrap replicates from the original dataset, with identical parameter conditions for phylogeny construction. Relative rate tests were performed using RRTree [12] under a Kimura 2-parameter substitution model with probabilities for rejecting the null hypothesis of rate equality calculated by the method of Li and Bousquet [13]. Branch length substitution base rate estimates were confirmed using the routine r8s [14] employing a penalized likelihood method. RESULTS An Updated M. domestica miRNA Map Screening MonDom5 with pre-miRNA sequences for chicken, mouse, and human from miRBase 10.0 identified 171 conserved miRNAs in the opossum genome including a few corrections of the previously reported data [7]. These microRNAs now encompass 107 single transcripts, 20 tandem loci, and six polycistronic loci. The complete, updated opossum miRNA file, with chromosome coordinates, is shown in miRBase style in Supplemental File 1 and a corrected and updated chromosome map indicating relative positions is shown in Supplemental File 2. In addition to adding 50 miRNAs to the opossum miRNA map that were not previously reported [7], there is one observation that merits specific comment. In miRBase, there are two miR-147 loci in human and chicken and one locus in the other species listed, including mouse and rat. When precursors for the miR-147s were screened in MonDom5, four loci were identified. These have been designated in Supplemental File 1 as mdo-miR147b1-4. We aligned the mdo-miR-147b1-4 pre-miRNAs plus a kilobase (kb) of upstream and downstream flanking sequence from MonDom 5 and observed sequence identities extending well beyond the pre-miRNAs. This is indicative of a larger duplication event but we were unable to detect any unequivocal tell-tale duplication identifiers such as flanking repeats. Inclusion of M. eugenii and O. anatinus Genomes Once validated, all of the opossum pre-miRNA sequences were used to screen the genome of the tammar wallaby in Trace Archives in GenBank, and of the platypus in Ensembl. Of the 171 sequences screened, 164 (95.9%) were found in at least one of the two genomes (Table 1). Overall, 136 (79.5%) were found in the wallaby and 147 (86.0%) were found in the platypus with 119 (69.6%) found in both genomes. It must be noted again that, since the current status of neither the wallaby nor the platypus genomes is as complete as the opossum genome, the comparative data presented here should not be considered complete for either species. It should also be noted that the seven miRNAs mapped in the opossum genome but not found in either the wallaby or platypus genomes are present in the chicken genome as well as in both mouse and human genomes. Thus, it is likely that these seven miRNAs will be found in other 24 The Open Genomics Journal, 2008, Volume 1 Devor and Peek Table 1. Conservation of Opossum MicroRNAs microRNA Chicken Platypus Wallaby