Antonio Marco

MicroRNA evolution by arm switching.

MicroRNAs (miRNAs) modulate transcript stability and translation. Functional mature miRNAs are processed from one or both arms of the hairpin precursor. The miR‐100/10 family has undergone three independent evolutionary events that have switched the arm from which the functional miRNA is processed. The dominant miR‐10 sequences in the insects Drosophila melanogaster and Tribolium castaneum are processed from opposite arms. However, the duplex produced by Dicer cleavage has an identical sequence in fly and beetle. Expression of the Tribolium miR‐10 sequence in Drosophila S2 cells recapitulates the native beetle pattern. Thus, arm usage is encoded in the primary miRNA sequence, but outside the mature miRNA duplex. We show that the predicted messenger RNA targets and inferred function of sequences from opposite arms differ significantly. Arm switching is likely to be general, and provides a fundamental mechanism to evolve the function of a miRNA locus and target gene network.

Functional Shifts in Insect microRNA Evolution

MicroRNAs (miRNAs) are short endogenous RNA molecules that regulate gene expression at the posttranscriptional level and have been shown to play critical roles during animal development. The identification and comparison of miRNAs in metazoan species are therefore paramount for our understanding of the evolution of body plans. We have characterized 203 miRNAs from the red flour beetle Tribolium castaneum by deep sequencing of small RNA libraries. We can conclude, from a single study, that the Tribolium miRNA set is at least 15% larger than that in the model insect Drosophila melanogaster (despite tens of high-throughput sequencing experiments in the latter). The rate of birth and death of miRNAs is high in insects. Only one-third of the Tribolium miRNA sequences are conserved in D. melanogaster, and at least 18 Tribolium miRNAs are conserved in vertebrates but lost in Drosophila. More than one-fifth of miRNAs that are conserved between Tribolium and Drosophila exhibit changes in the transcription, genomic organization, and processing patterns that lead to predicted functional shifts. For example, 13% of conserved miRNAs exhibit seed shifting, and we describe arm-switching events in 11% of orthologous pairs. These shifts fundamentally change the predicted targets and therefore function of orthologous miRNAs. In general, Tribolium miRNAs are more representative of the insect ancestor than Drosophila miRNAs and are more conserved in vertebrates.

Birds of a Feather: Neanderthal Exploitation of Raptors and Corvids

The hypothesis that Neanderthals exploited birds for the use of their feathers or claws as personal ornaments in symbolic behaviour is revolutionary as it assigns unprecedented cognitive abilities to these hominins. This inference, however, is based on modest faunal samples and thus may not represent a regular or systematic behaviour. Here we address this issue by looking for evidence of such behaviour across a large temporal and geographical framework. Our analyses try to answer four main questions: 1) does a Neanderthal to raptor-corvid connection exist at a large scale, thus avoiding associations that might be regarded as local in space or time?; 2) did Middle (associated with Neanderthals) and Upper Palaeolithic (associated with modern humans) sites contain a greater range of these species than Late Pleistocene paleontological sites?; 3) is there a taphonomic association between Neanderthals and corvids-raptors at Middle Palaeolithic sites on Gibraltar, specifically Gorhams, Vanguard and Ibex Caves? and; 4) was the extraction of wing feathers a local phenomenon exclusive to the Neanderthals at these sites or was it a geographically wider phenomenon?. We compiled a database of 1699 Pleistocene Palearctic sites based on fossil bird sites. We also compiled a taphonomical database from the Middle Palaeolithic assemblages of Gibraltar. We establish a clear, previously unknown and widespread, association between Neanderthals, raptors and corvids. We show that the association involved the direct intervention of Neanderthals on the bones of these birds, which we interpret as evidence of extraction of large flight feathers. The large number of bones, the variety of species processed and the different temporal periods when the behaviour is observed, indicate that this was a systematic, geographically and temporally broad, activity that the Neanderthals undertook. Our results, providing clear evidence that Neanderthal cognitive capacities were comparable to those of Modern Humans, constitute a major advance in the study of human evolution.

Evolution and function of the extended miR-2 microRNA family.

MicroRNAs are essential post-transcriptional regulators. Many animal microRNAs are clustered in the genome, and it has been shown that clustered microRNAs may be transcribed as a single transcript. Polycistronic microRNAs are often members of the same family, suggesting a role of tandem duplication in the emergence of clusters. The mir-2 microRNA family is the largest in Drosophila melanogaster, with 8 members that are mostly clustered in the genome. Previous studies suggest that the copy number and genomic distribution of mir-2 family members has been subject to significant change during evolution. The effects of such changes on their function are still unknown. Here we study the evolution of function in the mir-2 family. Our analyses show that, in spite of the change in number and organization among invertebrates, most mir-2 loci produce very similar mature microRNA products. Multiple mature miR-2 sequences are predicted to target genes involved in neural development in Drosophila. These targeting properties are conserved in the distant species Caenorhabditis elegans. Duplication followed by functional diversification is frequent during protein-coding gene evolution. However, our results suggest that the production of microRNA clusters by gene duplication rarely involves functional changes. This pattern of functional redundancy among clustered paralogous microRNAs reflects birth-and-death evolutionary dynamics. However, we identified a small number of mir-2 sequences in Drosophila that may have undergone functional shifts associated with genomic rearrangements. Therefore, redundancy in microRNA families may facilitate the acquisition of novel functional features.

Clusters of microRNAs emerge by new hairpins in existing transcripts

Genetic linkage may result in the expression of multiple products from a polycistronic transcript, under the control of a single promoter. In animals, protein-coding polycistronic transcripts are rare. However, microRNAs are frequently clustered in the genomes of animals, and these clusters are often transcribed as a single unit. The evolution of microRNA clusters has been the subject of much speculation, and a selective advantage of clusters of functionally related microRNAs is often proposed. However, the origin of microRNA clusters has not been so far explored. Here, we study the evolution of microRNA clusters in Drosophila melanogaster. We observed that the majority of microRNA clusters arose by the de novo formation of new microRNA-like hairpins in existing microRNA transcripts. Some clusters also emerged by tandem duplication of a single microRNA. Comparative genomics show that these clusters are unlikely to split or undergo rearrangements. We did not find any instances of clusters appearing by rearrangement of pre-existing microRNA genes. We propose a model for microRNA cluster evolution in which selection over one of the microRNAs in the cluster interferes with the evolution of the other linked microRNAs. Our analysis suggests that the study of microRNAs and small RNAs must consider linkage associations.

Positional conservation and amino acids shape the correct diagnosis and population frequencies of benign and damaging personal amino acid mutations

As the cost of DNA sequencing drops, we are moving beyond one genome per species to one genome per individual to improve prevention, diagnosis, and treatment of disease by using personal genotypes. Computational methods are frequently applied to predict impairment of gene function by nonsynonymous mutations in individual genomes and single nucleotide polymorphisms (nSNPs) in populations. These computational tools are, however, known to fail 15%-40% of the time. We find that accurate discrimination between benign and deleterious mutations is strongly influenced by the long-term (among species) history of positions that harbor those mutations. Successful prediction of known disease-associated mutations (DAMs) is much higher for evolutionarily conserved positions and for original-mutant amino acid pairs that are rarely seen among species. Prediction accuracies for nSNPs show opposite patterns, forecasting impediments to building diagnostic tools aiming to simultaneously reduce both false-positive and false-negative errors. The relative allele frequencies of mutations diagnosed as benign and damaging are predicted by positional evolutionary rates. These allele frequencies are modulated by the relative preponderance of the mutant allele in the set of amino acids found at homologous sites in other species (evolutionarily permissible alleles [EPAs]). The nSNPs found in EPAs are biochemically less severe than those missing from EPAs across all allele frequency categories. Therefore, it is important to consider position evolutionary rates and EPAs when interpreting the consequences and population frequencies of human mutations. The impending sequencing of thousands of human and many more vertebrate genomes will lead to more accurate classifiers needed in real-world applications.

Sex-biased expression of microRNAs in Schistosoma mansoni.

Schistosomiasis is an important neglected tropical disease caused by digenean helminth parasites of the genus Schistosoma. Schistosomes are unusual in that they are dioecious and the adult worms live in the blood system. MicroRNAs play crucial roles during gene regulation and are likely to be important in sex differentiation in dioecious species. Here we characterize 112 microRNAs from adult Schistosoma mansoni individuals, including 84 novel microRNA families, and investigate the expression pattern in different sexes. By deep sequencing, we measured the relative expression levels of conserved and newly identified microRNAs between male and female samples. We observed that 13 microRNAs exhibited sex-biased expression, 10 of which are more abundant in females than in males. Sex chromosomes showed a paucity of female-biased genes, as predicted by theoretical evolutionary models. We propose that the recent emergence of separate sexes in Schistosoma had an effect on the chromosomal distribution and evolution of microRNAs, and that microRNAs are likely to participate in the sex differentiation/maintenance process.

MicroRNAs from the same precursor have different targeting properties

BackgroundThe processing of a microRNA results in an intermediate duplex of two potential mature products that derive from the two arms (5′ and 3′) of the precursor hairpin. It is often suggested that one of the sequences is degraded and the other is incorporated into the RNA-induced silencing complex. However, both precursor arms may give rise to functional levels of mature microRNA and the dominant product may change from species to species, from tissue to tissue, or between developmental stages. Therefore, both arms of the precursor have the potential to produce functional mature microRNAs.ResultsWe have investigated the relationship between predicted mRNA targets of mature sequences derived from the 5′ and 3′ arms of the same pre-microRNAs. Using six state-of-the-art target prediction algorithms, we find that 5′/3′ microRNA pairs target different sites in 3′ untranslated regions of mRNAs. We also find that these pairs do not generally target overlapping sets of genes, or functionally related genes.ConclusionsWe show that alternative mature products produced from the same precursor microRNAs have different targeting properties and therefore different biological functions. These data strongly suggest that developmental or evolutionary changes in arm choice will have significant functional consequences.

Relationship between gene co-expression and sharing of transcription factor binding sites in Drosophila melanogaster

MOTIVATION In functional genomics, it is frequently useful to correlate expression levels of genes to identify transcription factor binding sites (TFBS) via the presence of common sequence motifs. The underlying assumption is that co-expressed genes are more likely to contain shared TFBS and, thus, TFBS can be identified computationally. Indeed, gene pairs with a very high expression correlation show a significant excess of shared binding sites in yeast. We have tested this assumption in a more complex organism, Drosophila melanogaster, by using experimentally determined TFBS and microarray expression data. We have also examined the reverse relationship between the expression correlation and the extent of TFBS sharing. RESULTS Pairs of genes with shared TFBS show, on average, a higher degree of co-expression than those with no common TFBS in Drosophila. However, the reverse does not hold true: gene pairs with high expression correlations do not share significantly larger numbers of TFBS. Exception to this observation exists when comparing expression of genes from the earliest stages of embryonic development. Interestingly, semantic similarity between gene annotations (Biological Process) is much better associated with TFBS sharing, as compared to the expression correlation. We discuss these results in light of reverse engineering approaches to computationally predict regulatory sequences by using comparative genomics.

The earliest pigeon fanciers

Feral Pigeons have colonised all corners of the Earth, having developed a close association with humans and their activities. The wild ancestor of the Feral Pigeon, the Rock Dove, is a species of rocky habitats, nesting typically on cliff ledges and at the entrance to large caves. This habit would have brought them into close contact with cave-dwelling humans, a relationship usually linked to the development of dwellings in the Neolithic. We show that the association between humans and Rock Doves is an ancient one with its roots in the Palaeolithic and predates the arrival of modern humans into Europe. At Gorhams Cave, Gibraltar, the Neanderthals exploited Rock Doves for food for a period of over 40 thousand years, the earliest evidence dating to at least 67 thousand years ago. We show that the exploitation was not casual or sporadic, having found repeated evidence of the practice in different, widely spaced, temporal contexts within the cave. Our results point to hitherto unappreciated capacities of the Neanderthals to exploit birds as food resources on a regular basis. More so, they were practising it long before the arrival of modern humans and had therefore invented it independently.