Ana C. Marques

Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

A finished clone-based assembly of the mouse genome reveals extensive recent sequence duplication during recent evolution and rodent-specific expansion of certain gene families. Newly assembled duplications contain protein-coding genes that are mostly involved in reproductive function.

A Transcriptomic Atlas of Mouse Neocortical Layers

Summary In the mammalian cortex, neurons and glia form a patterned structure across six layers whose complex cytoarchitectonic arrangement is likely to contribute to cognition. We sequenced transcriptomes from layers 1-6b of different areas (primary and secondary) of the adult (postnatal day 56) mouse somatosensory cortex to understand the transcriptional levels and functional repertoires of coding and noncoding loci for cells constituting these layers. A total of 5,835 protein-coding genes and 66 noncoding RNA loci are differentially expressed (“patterned”) across the layers, on the basis of a machine-learning model (naive Bayes) approach. Layers 2-6b are each associated with specific functional and disease annotations that provide insights into their biological roles. This new resource (http://genserv.anat.ox.ac.uk/layers) greatly extends currently available resources, such as the Allen Mouse Brain Atlas and microarray data sets, by providing quantitative expression levels, by being genome-wide, by including novel loci, and by identifying candidate alternatively spliced transcripts that are differentially expressed across layers.

Rapid turnover of long noncoding RNAs and the evolution of gene expression.

A large proportion of functional sequence within mammalian genomes falls outside protein-coding exons and can be transcribed into long RNAs. However, the roles in mammalian biology of long noncoding RNA (lncRNA) are not well understood. Few lncRNAs have experimentally determined roles, with some of these being lineage-specific. Determining the extent by which transcription of lncRNA loci is retained or lost across multiple evolutionary lineages is essential if we are to understand their contribution to mammalian biology and to lineage-specific traits. Here, we experimentally investigated the conservation of lncRNA expression among closely related rodent species, allowing the evolution of DNA sequence to be uncoupled from evolution of transcript expression. We generated total RNA (RNAseq) and H3K4me3-bound (ChIPseq) DNA data, and combined both to construct catalogues of transcripts expressed in the adult liver of Mus musculus domesticus (C57BL/6J), Mus musculus castaneus, and Rattus norvegicus. We estimated the rate of transcriptional turnover of lncRNAs and investigated the effects of their lineage-specific birth or death. LncRNA transcription showed considerably greater gain and loss during rodent evolution, compared with protein-coding genes. Nucleotide substitution rates were found to mirror the in vivo transcriptional conservation of intergenic lncRNAs between rodents: only the sequences of noncoding loci with conserved transcription were constrained. Finally, we found that lineage-specific intergenic lncRNAs appear to be associated with modestly elevated expression of genomically neighbouring protein-coding genes. Our findings show that nearly half of intergenic lncRNA loci have been gained or lost since the last common ancestor of mouse and rat, and they predict that such rapid transcriptional turnover contributes to the evolution of tissue- and lineage-specific gene expression.

Catalogues of mammalian long noncoding RNAs: modest conservation and incompleteness.

BackgroundDespite increasing interest in the noncoding fraction of transcriptomes, the number, species-conservation and functions, if any, of many non-protein-coding transcripts remain to be discovered. Two extensive long intergenic noncoding RNA (ncRNA) transcript catalogues are now available for mouse: over 3,000 macroRNAs identified by cDNA sequencing, and 1,600 long intergenic noncoding RNA (lincRNA) intervals that are predicted from chromatin-state maps. Previously we showed that macroRNAs tend to be more highly conserved than putatively neutral sequence, although only 5% of bases are predicted as constrained. By contrast, over a thousand lincRNAs were reported as being highly conserved. This apparent difference may account for the surprisingly small fraction (11%) of transcripts that are represented in both catalogues. Here we sought to resolve the reported discrepancy between the evolutionary rates for these two sets.ResultsOur analyses reveal lincRNA and macroRNA exon sequences to be subject to the same relatively low degree of sequence constraint. Nonetheless, our observations are consistent with the functionality of a fraction of ncRNA in these sets, with up to a quarter of ncRNA exons having evolved significantly slower than neighboring neutral sequence. The more tissue-specific macroRNAs are enriched in predicted RNA secondary structures and thus may often act in trans, whereas the more highly and broadly expressed lincRNAs appear more likely to act in the cis-regulation of adjacent transcription factor genes.ConclusionsTaken together, our results indicate that each of the two ncRNA catalogues unevenly and lightly samples the true, much larger, ncRNA repertoire of the mouse.

Chromosomal gene movements reflect the recent origin and biology of therian sex chromosomes

Mammalian sex chromosomes stem from ancestral autosomes and have substantially differentiated. It was shown that X-linked genes have generated duplicate intronless gene copies (retrogenes) on autosomes due to this differentiation. However, the precise driving forces for this out-of-X gene “movement” and its evolutionary onset are not known. Based on expression analyses of male germ-cell populations, we here substantiate and extend the hypothesis that autosomal retrogenes functionally compensate for the silencing of their X-linked housekeeping parental genes during, but also after, male meiotic sex chromosome inactivation (MSCI). Thus, sexually antagonistic forces have not played a major role for the selective fixation of X-derived gene copies in mammals. Our dating analyses reveal that although retrogenes were produced ever since the common mammalian ancestor, selectively driven retrogene export from the X only started later, on the placental mammal (eutherian) and marsupial (metatherian) lineages, respectively. Together, these observations suggest that chromosome-wide MSCI emerged close to the eutherian–marsupial split approximately 180 million years ago. Given that MSCI probably reflects the spread of the recombination barrier between the X and Y, crucial for their differentiation, our data imply that these chromosomes became more widely differentiated only late in the therian ancestor, well after the divergence of the monotreme lineage. Thus, our study also provides strong independent support for the recent notion that our sex chromosomes emerged, not in the common ancestor of all mammals, but rather in the therian ancestor, and therefore are much younger than previously thought.

Identification and properties of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome.

The functional repertoire of long intergenic noncoding RNA (lincRNA) molecules has begun to be elucidated in mammals. Determining the biological relevance and potential gene regulatory mechanisms of these enigmatic molecules would be expedited in a more tractable model organism, such as Drosophila melanogaster. To this end, we defined a set of 1,119 putative lincRNA genes in D. melanogaster using modENCODE whole transcriptome (RNA-seq) data. A large majority (1.1 of 1.3 Mb; 85%) of these bases were not previously reported by modENCODE as being transcribed. Significant selective constraint on the sequences of these loci predicts that virtually all have sustained functionality across the Drosophila clade. We observe biases in lincRNA genomic locations and expression profiles that are consistent with some of these lincRNAs being involved in the regulation of neighboring protein-coding genes with developmental functions. We identify lincRNAs that may be important in the developing nervous system and in male-specific organs, such as the testes. LincRNA loci were also identified whose positions, relative to nearby protein-coding loci, are equivalent between D. melanogaster and mouse. This study predicts that the genomes of not only vertebrates, such as mammals, but also an invertebrate (fruit fly) harbor large numbers of lincRNA loci. Our findings now permit exploitation of Drosophila genetics for the investigation of lincRNA mechanisms, including lincRNAs with potential functional analogues in mammals.

Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs

BackgroundMammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers.ResultsWe integrated high-resolution maps of transcriptional initiation and transcription to annotate a conservative set of intergenic lncRNAs expressed in mouse erythroblasts. We subclassified intergenic lncRNAs according to chromatin status at transcriptional initiation regions, defined by relative levels of histone H3K4 mono- and trimethylation. These transcripts are almost evenly divided between those arising from enhancer-associated (elncRNA) or promoter-associated (plncRNA) elements. These two classes of 5′ capped and polyadenylated RNA transcripts are indistinguishable with regard to their length, number of exons or transcriptional orientation relative to their closest neighboring gene. Nevertheless, elncRNAs are more tissue-restricted, less highly expressed and less well conserved during evolution. Of considerable interest, we found that expression of elncRNAs, but not plncRNAs, is associated with enhanced expression of neighboring protein-coding genes during erythropoiesis.ConclusionsWe have determined globally the sites of initiation of intergenic lncRNAs in erythroid cells, allowing us to distinguish two similarly abundant classes of transcripts. Different correlations between the levels of elncRNAs, plncRNAs and expression of neighboring genes suggest that functional lncRNAs from the two classes may play contrasting roles in regulating the transcript abundance of local or distal loci.

Birth and Rapid Subcellular Adaptation of a Hominoid-Specific CDC14 Protein

Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated) and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18–25 million years ago (Mya). CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7–12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them) to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor ∼18–25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Intergenic lncRNAs and the evolution of gene expression

Eukaryote genomes encode a surprisingly large number of noncoding transcripts. Around two-thirds of human transcribed loci do not encode protein, and many are intergenic and produce long (>200 nucleotides) noncoding RNAs (lncRNAs). Extensive analyses using comparative genomics and transcriptomics approaches have established that lncRNA sequence and transcription tend to turn over rapidly during evolution. Our appreciation of the biological roles of lncRNAs, based only on a handful of transcripts with well-characterized functions, is that lncRNAs have diverse roles in regulating gene expression. These proposed roles together with their rapid rates of evolution suggest that lncRNAs could contribute to the divergent expression patterns observed among species and potentially to the origin of new traits.