Conserved microRNA editing in mammalian evolution, development and disease.

BACKGROUND: Mammalian microRNAs (miRNAs) are sometimes subject to adenosine-to-inosine RNA editing, which can lead to dramatic changes in miRNA target specificity or expression levels. However, although a few miRNAs are known to be edited at identical positions in human and mouse, the evolution of miRNA editing has not been investigated in detail. In this study, we identify conserved miRNA editing events in a range of mammalian and non-mammalian species. RESULTS: We demonstrate deep conservation of several site-specific miRNA editing events, including two that date back to the common ancestor of mammals and bony fishes some 450 million years ago. We also find evidence of a recent expansion of an edited miRNA family in placental mammals and show that editing of these miRNAs is associated with changes in target mRNA expression during primate development and aging. While global patterns of miRNA editing tend to be conserved across species, we observe substantial variation in editing frequencies depending on tissue, age and disease state: editing is more frequent in neural tissues compared to heart, kidney and testis; in older compared to younger individuals; and in samples from healthy tissues compared to tumors, which together suggests that miRNA editing might be associated with a reduced rate of cell proliferation. CONCLUSIONS: Our results show that site-specific miRNA editing is an evolutionarily conserved mechanism, which increases the functional diversity of mammalian miRNA transcriptomes. Furthermore, we find that although miRNA editing is rare compared to editing of long RNAs, miRNAs are greatly overrepresented among conserved editing targets

Repurposing of promoters and enhancers during mammalian evolution.

Promoters and enhancers-key controllers of gene expression-have long been distinguished from each other based on their function. However, recent work suggested that common architectural and functional features might have facilitated the conversion of one type of element into the other during evolution. Here, based on cross-mammalian analyses of epigenome and transcriptome data, we provide support for this hypothesis by detecting 445 regulatory elements with signatures of activity turnover (termed P/E elements). Most events represent transformations of putative ancestral enhancers into promoters, leading to the emergence of species-specific transcribed loci or 5' exons. Distinct GC sequence compositions and stabilizing 5' splicing (U1) regulatory motif patterns may have predisposed P/E elements to regulatory repurposing, and changes in the U1 and polyadenylation signal densities and distributions likely drove the evolutionary activity switches. Our work suggests that regulatory repurposing facilitated regulatory innovation and the origination of new genes and exons during evolution

ProteinHistorian: Tools for the Comparative Analysis of Eukaryote Protein Origin

The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are freely available under the GNU public license at http://lighthouse.ucsf.edu/ProteinHistorian/

A Selection Index for Gene Expression Evolution and Its Application to the Divergence between Humans and Chimpanzees

The importance of gene regulation in animal evolution is a matter of long-standing interest, but measuring the impact of selection on gene expression has proven a challenge. Here, we propose a selection index of gene expression as a straightforward method for assessing the mode and strength of selection operating on gene expression levels. The index is based on the widely used McDonald-Kreitman test and requires the estimation of four quantities: the within-species and between-species expression variances as well as the sequence heterozygosity and divergence of neutrally evolving sequences. We apply the method to data from human and chimpanzee lymphoblastoid cell lines and show that gene expression is in general under strong stabilizing selection. We also demonstrate how the same framework can be used to estimate the proportion of adaptive gene expression evolution

A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme

Editing in microRNAs, particularly in seed can significantly alter the choice of their target genes. We show that out of 13 different human tissues, different regions of brain showed higher adenosine to inosine (A-to-I) editing in mature miRNAs. These events were enriched in seed sequence (73.33%), which was not observed for cytosine to uracil (17.86%) editing. More than half of the edited miRNAs showed increased stability, 72.7% of which had ΔΔG values less than -6.0 Kcal/mole and for all of them the edited adenosines mis-paired with cytosines on the pre-miRNA structure. A seed-editing event in hsa-miR-411 (with A – C mismatch) lead to increased expression of the mature form compared to the unedited version in cell culture experiments. Further, small RNA sequencing of GBM patient identified significant miRNA hypoediting which correlated with downregulation of ADAR2 both in metadata and qRT-PCR based validation. Twenty-two significant (11 novel) A-to-I hypoediting events were identified in GBM samples. This study highlights the importance of specific sequence and structural requirements of pre-miRNA for editing along with a suggestive crucial role for ADAR2. Enrichment of A-to-I editing in seed sequence highlights this as an important layer for genomic regulation in health and disease, especially in human brain

Germ Cell-Specific Targeting of DICER or DGCR8 Reveals a Novel Role for Endo-siRNAs in the Progression of Mammalian Spermatogenesis and Male Fertility

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process

The life history of retrocopies illuminates the evolution of new mammalian genes.

New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution

Transposable Elements: Insertion Pattern and Impact on Gene Expression Evolution in Hominids

Transposable elements (TEs) can affect the regulation of nearby genes through several mechanisms. Here, we examine to what extent recent TE insertions have contributed to the evolution of gene expression in hominids. We compare expression levels of human and chimpanzee orthologs and detect a weak increase in expression divergence (ED) for genes with species-specific TE insertions compared with unaffected genes. However, we show that genes with TE insertions predating the human-chimpanzee split also exhibit a similar increase in ED and therefore conclude that the increase is not due to the transcriptional influence of the TEs. These results are further confirmed by lineage-specific analysis of ED, using rhesus macaque as an outgroup: Human-chimpanzee ortholog pairs, where one ortholog has suffered TE insertion but not the other, do not show increased ED along the lineage where the insertion occurred, relative to the other lineage. We also show that genes with recent TE insertions tend to produce more alternative transcripts but find no evidence that the TEs themselves promote transcript diversity. Finally, we observe that TEs are enriched upstream relative to downstream of genes and show that this is due to insertional bias, rather than selection, because this bias is only observed in genes expressed in the germ line. This provides an alternative neutral explanation for the accumulation of TEs in upstream sequences