Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii.

BACKGROUND: Restorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered. RESULTS: We generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process. CONCLUSIONS: In this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

An integrated model for termination of RNA polymerase III transcription.

RNA polymerase III (RNAPIII) synthesizes essential and abundant noncoding RNAs such as transfer RNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the nontemplate strand. Here, we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

Enhancer-gene maps in the human and zebrafish genomes using evolutionary linkage conservation.

The spatiotemporal expression of genes is controlled by enhancer sequences that bind transcription factors. Identifying the target genes of enhancers remains difficult because enhancers regulate gene expression over long genomic distances. To address this, we used an evolutionary approach to build two genome-wide maps of predicted enhancer-gene associations in the human and zebrafish genomes. Evolutionary conserved sequences were linked to their predicted target genes using PEGASUS, a bioinformatics method that relies on evolutionary conservation of synteny. The analysis of these maps revealed that the number of predicted enhancers linked to a gene correlate with its expression breadth. Comparison of both maps identified hundreds of putative vertebrate ancestral regulatory relationships from which we could determine that predicted enhancer-gene distances scale with genome size despite strong positional conservation. The two maps represent a resource for further studies, including the prioritization of sequence variants in whole genome sequence of patients affected by genetic diseases.

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Evolutionary forces affecting synonymous variations in plant genomes.

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones.

The bimodal distribution of genic GC content is ancestral to monocot species.

In grasses such as rice or maize, the distribution of genic GC content is well known to be bimodal. It is mainly driven by GC content at third codon positions (GC3 for short). This feature is thought to be specific to grasses as closely related species like banana have a unimodal GC3 distribution. GC3 is associated with numerous genomics features and uncovering the origin of this peculiar distribution will help understanding the potential roles and consequences of GC3 variations within and between genomes. Until recently, the origin of the peculiar GC3 distribution in grasses has remained unknown. Thanks to the recent publication of several complete genomes and transcriptomes of nongrass monocots, we studied more than 1,000 groups of one-to-one orthologous genes in seven grasses and three outgroup species (banana, palm tree, and yam). Using a maximum likelihood-based method, we reconstructed GC3 at several ancestral nodes. We found that the bimodal GC3 distribution observed in extant grasses is ancestral to both grasses and most monocot species, and that other species studied here have lost this peculiar structure. We also found that GC3 in grass lineages is globally evolving very slowly and that the decreasing GC3 gradient observed from 5' to 3' along coding sequences is also conserved and ancestral to monocots. This result strongly challenges the previous views on the specificity of grass genomes and we discuss its implications for the possible causes of the evolution of GC content in monocots.

GC content evolution in coding regions of angiosperm genomes: a unifying hypothesis.

In angiosperms (as in other species), GC content varies along and between genes, within a genome, and between genomes of different species, but the reason for this distribution is still an open question. Grass genomes are particularly intriguing because they exhibit a strong bimodal distribution of genic GC content and a sharp 5'-3' decreasing GC content gradient along most genes. Here, we propose a unifying model to explain the main patterns of GC content variation at the gene and genome scale. We argue that GC content patterns could be mainly determined by the interactions between gene structure, recombination patterns, and GC-biased gene conversion. Recent studies on fine-scale recombination maps in angiosperms support this hypothesis and previous results also fit this model. We propose that our model could be used as a null hypothesis to search for additional forces that affect GC content in angiosperms.

Meiotic recombination strongly influences GC-content evolution in short regions in the mouse genome.

Meiotic recombination is known to influence GC-content evolution in large regions of mammalian genomes by favoring the fixation of G and C alleles and increasing the rate of A/T to G/C substitutions. This process is known as GC-biased gene conversion (gBGC). Until recently, genome-wide measures of fine-scale recombination activity were unavailable in mice. Additionally, comparative studies focusing on mouse were limited as the closest organism with its genome fully sequenced was rat. Here, we make use of the recent mapping of double strand breaks (DSBs), the first step of meiotic recombination, in the mouse genome and of the sequencing of mouse closely related subspecies to analyze the fine-scale evolutionary signature of meiotic recombination on GC-content evolution in recombination hotspots, short regions that undergo extreme rates of recombination. We measure substitution rates around DSB hotspots and observe that gBGC is affecting a very short region (≈ 1 kbp) in length around these hotspots. Furthermore, we can infer that the locations of hotspots evolved rapidly during mouse evolution.

Substitution patterns are under different influences in primates and rodents.

There are large-scale variations of the GC-content along mammalian chromosomes that have been called isochore structures. Primates and rodents have different isochore structures, which suggests that these lineages exhibit different modes of GC-content evolution. It has been shown that, in the human lineage, GC-biased gene conversion (gBGC), a neutral process associated with meiotic recombination, acts on GC-content evolution by influencing A or T to G or C substitution rates. We computed genome-wide substitution patterns in the mouse lineage from multiple alignments and compared them with substitution patterns in the human lineage. We found that in the mouse lineage, gBGC is active but weaker than in the human lineage and that male-specific recombination better predicts GC-content evolution than female-specific recombination. Furthermore, we were able to show that G or C to A or T substitution rates are predicted by a combination of different factors in both lineages. A or T to G or C substitution rates are most strongly predicted by meiotic recombination in the human lineage but by CpG odds ratio (the observed CpG frequency normalized by the expected CpG frequency) in the mouse lineage, suggesting that substitution patterns are under different influences in primates and rodents.

Does lack of recombination enhance asymmetric evolution among duplicate genes? Insights from the Drosophila melanogaster genome.

Gene duplication has different outcomes: pseudogenization (death of one of the two copies), gene amplification (both copies remain the same), sub-functionalization (both copies are required to perform the ancestral function) and neo-functionalization (one copy acquires a new function). Asymmetric evolution (one copy evolves faster than the other) is usually seen as a signature of neo-functionalization. However, it has been proposed that sub-functionalization could also generate asymmetric evolution among duplicate genes when they experience different local recombination rates. Indeed, the low recombination copy is expected to evolve faster because of Hill-Robertson effects. Here we tested this idea with about 100 pairs of young duplicates from the Drosophila melanogaster genome. Looking only at young duplicates allowed us to compare recombination rates and evolutionary rates on a similar time-scale contrary to previous work. We found that dispersed pairs tend to evolve more asymmetrically than tandem ones. Among dispersed copies, the low recombination copy tends to be the fast-evolving one. We also tested the possibility that all this was explained by a confounding factor (expression level) but found no evidence for it. In conclusion, our results do support the idea that asymmetric evolution among duplicates is enhanced by restricted recombination. However, further work is needed to clearly distinguish between sub-functionalization and neo-functionalization for the asymmetrically-evolving duplicate pairs that we found.