Evolution and Phylogeny of MicroRNAs - Protocols, Pitfalls, and Problems.

MicroRNAs are important regulators in many eukaryotic lineages. Typical miRNAs have a length of about 22nt and are processed from precursors that form a characteristic hairpin structure. Once they appear in a genome, miRNAs are among the best-conserved elements in both animal and plant genomes. Functionally, they play an important role in particular in development. In contrast to protein-coding genes, miRNAs frequently emerge de novo. The genomes of animals and plants harbor hundreds of mutually unrelated families of homologous miRNAs that tend to be persistent throughout evolution. The evolution of their genomic miRNA complement closely correlates with important morphological innovation. In addition, miRNAs have been used as valuable characters in phylogenetic studies. An accurate and comprehensive annotation of miRNAs is required as a basis to understand their impact on phenotypic evolution. Since experimental data on miRNA expression are limited to relatively few species and are subject to unavoidable ascertainment biases, it is inevitable to complement miRNA sequencing by homology based annotation methods. This chapter reviews the state of the art workflows for homology based miRNA annotation, with an emphasis on their limitations and open problems.

The Genome of the "Sea Vomit" Didemnum vexillum.

Tunicates are the sister group of vertebrates and thus occupy a key position for investigations into vertebrate innovations as well as into the consequences of the vertebrate-specific genome duplications. Nevertheless, tunicate genomes have not been studied extensively in the past, and comparative studies of tunicate genomes have remained scarce. The carpet sea squirt Didemnum vexillum, commonly known as "sea vomit", is a colonial tunicate considered an invasive species with substantial ecological and economical risk. We report the assembly of the D. vexillum genome using a hybrid approach that combines 28.5 Gb Illumina and 12.35 Gb of PacBio data. The new hybrid scaffolded assembly has a total size of 517.55 Mb that increases contig length about eightfold compared to previous, Illumina-only assembly. As a consequence of an unusually high genetic diversity of the colonies and the moderate length of the PacBio reads, presumably caused by the unusually acidic milieu of the tunic, the assembly is highly fragmented (L50 = 25,284, N50 = 6539). It is sufficient, however, for comprehensive annotations of both protein-coding genes and non-coding RNAs. Despite its shortcomings, the draft assembly of the "sea vomit" genome provides a valuable resource for comparative tunicate genomics and for the study of the specific properties of colonial ascidians.

miRNAture-Computational Detection of microRNA Candidates.

Homology-based annotation of short RNAs, including microRNAs, is a difficult problem because their inherently small size limits the available information. Highly sensitive methods, including parameter optimized blast, nhmmer, or cmsearch runs designed to increase sensitivity inevitable lead to large numbers of false positives, which can be detected only by detailed analysis of specific features typical for a RNA family and/or the analysis of conservation patterns in structure-annotated multiple sequence alignments. The miRNAture pipeline implements a workflow specific to animal microRNAs that automatizes homology search and validation steps. The miRNAture pipeline yields very good results for a large number of "typical" miRBase families. However, it also highlights difficulties with atypical cases, in particular microRNAs deriving from repetitive elements and microRNAs with unusual, branched precursor structures and atypical locations of the mature product, which require specific curation by domain experts.

Nonprotein-Coding RNAs as Regulators of Development in Tunicates.

Tunicates, or urochordates, are a group of small marine organisms that are found widely throughout the seas of the world. As most plausible sister group of the vertebrates, they are of utmost importance for a comprehensive understanding of chordate evolution; hence, they have served as model organisms for many aspects of the developmental biology. Current genomic analysis of tunicates indicates that their genomes evolved with a fast rate not only at the level of nucleotide substitutions but also in terms of genomic organization. The latter involves genome reduction, rearrangements, as well as the loss of some important coding and noncoding RNA (ncRNAs) elements and even entire genomic regions that are otherwise well conserved. These observations are largely based on evidence from comparative genomics resulting from the analysis of well-studied gene families such as the Hox genes and their noncoding elements. In this chapter, the focus lies on the ncRNA complement of tunicates, with a particular emphasis on microRNAs, which have already been studied extensively for other animal clades. MicroRNAs are known as important regulators of key genes in animal development, and they are intimately related to the increase morphological complexity in higher metazoans. Here we review the discovery, evolution, and genome organization of the miRNA repertoire, which has been drastically reduced and restructured in tunicates compared to the chordate ancestor. Known functions of microRNAs as regulators of development in tunicates are a central topic. For instance, we consider the role of miRNAs as regulators of the muscle development and their importance in the regulation of the differential expression during the oral siphon regeneration. Beyond microRNAs, we touch upon the functions of some other ncRNAs such as yellow crescent RNA, moRNAs, RMST lncRNAs, or spliced-leader (SL) RNAs, which have diverse functions associated with the embryonic development, neurogenesis, and mediation of mRNA stability in general.

Automated detection of ncRNAs in the draft genome sequence of a colonial tunicate: the carpet sea squirt Didemnum vexillum.

BACKGROUND: The colonial ascidian Didemnum vexillum, sea carpet squirt, is not only a key marine organism to study morphological ancestral patterns of chordates evolution but it is also of great ecological importance due to its status as a major invasive species. Non-coding RNAs, in particular microRNAs (miRNAs), are important regulatory genes that impact development and environmental adaptation. Beyond miRNAs, not much in known about tunicate ncRNAs. RESULTS: We provide here a comprehensive homology-based annotation of non-coding RNAs in the recently sequenced genome of D. vexillum. To this end we employed a combination of several computational approaches, including blast searches with a wide range of parameters, and secondary structured centered survey with infernal. The resulting candidate set was curated extensively to produce a high-quality ncRNA annotation of the first draft of the D. vexillum genome. It comprises 57 miRNA families, 4 families of ribosomal RNAs, 22 isoacceptor classes of tRNAs (of which more than 72 % of loci are pseudogenes), 13 snRNAs, 12 snoRNAs, and 1 other RNA family. Additionally, 21 families of mitochondrial tRNAs and 2 of mitochondrial ribosomal RNAs and 1 long non-coding RNA. CONCLUSIONS: The comprehensive annotation of the D. vexillum non-coding RNAs provides a starting point towards a better understanding of the restructuring of the small RNA system in ascidians. Furthermore it provides a valuable research for efforts to establish detailed non-coding RNA annotations for other recently published and recently sequences in tunicate genomes.

Orthologs, turn-over, and remolding of tRNAs in primates and fruit flies.

BACKGROUND: Transfer RNAs (tRNAs) are ubiquitous in all living organism. They implement the genetic code so that most genomes contain distinct tRNAs for almost all 61 codons. They behave similar to mobile elements and proliferate in genomes spawning both local and non-local copies. Most tRNA families are therefore typically present as multicopy genes. The members of the individual tRNA families evolve under concerted or rapid birth-death evolution, so that paralogous copies maintain almost identical sequences over long evolutionary time-scales. To a good approximation these are functionally equivalent. Individual tRNA copies thus are evolutionary unstable and easily turn into pseudogenes and disappear. This leads to a rapid turnover of tRNAs and often large differences in the tRNA complements of closely related species. Since tRNA paralogs are not distinguished by sequence, common methods cannot not be used to establish orthology between tRNA genes. RESULTS: In this contribution we introduce a general framework to distinguish orthologs and paralogs in gene families that are subject to concerted evolution. It is based on the use of uniquely aligned adjacent sequence elements as anchors to establish syntenic conservation of sequence intervals. In practice, anchors and intervals can be extracted from genome-wide multiple sequence alignments. Syntenic clusters of concertedly evolving genes of different families can then be subdivided by list alignments, leading to usually small clusters of candidate co-orthologs. On the basis of recent advances in phylogenetic combinatorics, these candidate clusters can be further processed by cograph editing to recover their duplication histories. We developed a workflow that can be conceptualized as stepwise refinement of a graph of homologous genes. We apply this analysis strategy with different types of synteny anchors to investigate the evolution of tRNAs in primates and fruit flies. We identified a large number of tRNA remolding events concentrated at the tips of the phylogeny. With one notable exception all phylogenetically old tRNA remoldings do not change the isoacceptor class. CONCLUSIONS: Gene families evolving under concerted evolution are not amenable to classical phylogenetic analyses since paralogs maintain identical, species-specific sequences, precluding the estimation of correct gene trees from sequence differences. This leaves conservation of syntenic arrangements with respect to "anchor elements" that are not subject to concerted evolution as the only viable source of phylogenetic information. We have demonstrated here that a purely synteny-based analysis of tRNA gene histories is indeed feasible. Although the choice of synteny anchors influences the resolution in particular when tight gene clusters are present, and the quality of sequence alignments, genome assemblies, and genome rearrangements limits the scope of the analysis, largely coherent results can be obtained for tRNAs. In particular, we conclude that a large fraction of the tRNAs are recent copies. This proliferation is compensated by rapid pseudogenization as exemplified by many very recent alloacceptor remoldings.