Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage.

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

Piwi Is Required during Drosophila Embryogenesis to License Dual-Strand piRNA Clusters for Transposon Repression in Adult Ovaries.

Most piRNAs in the Drosophila female germline are transcribed from heterochromatic regions called dual-strand piRNA clusters. Histone 3 lysine 9 trimethylation (H3K9me3) is required for licensing piRNA production by these clusters. However, it is unclear when and how they acquire this permissive heterochromatic state. Here, we show that transient Piwi depletion in Drosophila embryos results in H3K9me3 decrease at piRNA clusters in ovaries. This is accompanied by impaired biogenesis of ovarian piRNAs, accumulation of transposable element transcripts, and female sterility. Conversely, Piwi depletion at later developmental stages does not disturb piRNA cluster licensing. These results indicate that the identity of piRNA clusters is epigenetically acquired in a Piwi-dependent manner during embryonic development, which is reminiscent of the widespread genome reprogramming occurring during early mammalian zygotic development.

microRNA target prediction programs predict many false positives.

According to the current view, each microRNA regulates hundreds of genes. Computational tools aim at identifying microRNA targets, usually selecting evolutionarily conserved microRNA binding sites. While the false positive rates have been evaluated for some prediction programs, that information is rarely put forward in studies making use of their predictions. Here, we provide evidence that such predictions are often biologically irrelevant. Focusing on miR-223-guided repression, we observed that it is often smaller than inter-individual variability in gene expression among wild-type mice, suggesting that most predicted targets are functionally insensitive to that microRNA. Furthermore, we found that human haplo-insufficient genes tend to bear the most highly conserved microRNA binding sites. It thus appears that biological functionality of microRNA binding sites depends on the dose-sensitivity of their host gene and that, conversely, it is unlikely that every predicted microRNA target is dose-sensitive enough to be functionally regulated by microRNAs. We also observed that some mRNAs can efficiently titrate microRNAs, providing a reason for microRNA binding site conservation for inefficiently repressed targets. Finally, many conserved microRNA binding sites are conserved in a microRNA-independent fashion: Sequence elements may be conserved for other reasons, while being fortuitously complementary to microRNAs. Collectively, our data suggest that the role of microRNAs in normal and pathological conditions has been overestimated due to the frequent overlooking of false positive rates.

The somatic piRNA pathway controls germline transposition over generations.

Transposable elements (TEs) are parasitic DNA sequences that threaten genome integrity by replicative transposition in host gonads. The Piwi-interacting RNAs (piRNAs) pathway is assumed to maintain Drosophila genome homeostasis by downregulating transcriptional and post-transcriptional TE expression in the ovary. However, the bursts of transposition that are expected to follow transposome derepression after piRNA pathway impairment have not yet been reported. Here, we show, at a genome-wide level, that piRNA loss in the ovarian somatic cells boosts several families of the endogenous retroviral subclass of TEs, at various steps of their replication cycle, from somatic transcription to germinal genome invasion. For some of these TEs, the derepression caused by the loss of piRNAs is backed up by another small RNA pathway (siRNAs) operating in somatic tissues at the post transcriptional level. Derepressed transposition during 70 successive generations of piRNA loss exponentially increases the genomic copy number by up to 10-fold.

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Compositional biases among synonymous substitutions cause conflict between gene and protein trees for plastid origins.

Archaeplastida (=Kingdom Plantae) are primary plastid-bearing organisms that evolved via the endosymbiotic association of a heterotrophic eukaryote host cell and a cyanobacterial endosymbiont approximately 1,400 Ma. Here, we present analyses of cyanobacterial and plastid genomes that show strongly conflicting phylogenies based on 75 plastid (or nuclear plastid-targeted) protein-coding genes and their direct translations to proteins. The conflict between genes and proteins is largely robust to the use of sophisticated data- and tree-heterogeneous composition models. However, by using nucleotide ambiguity codes to eliminate synonymous substitutions due to codon-degeneracy, we identify a composition bias, and dependent codon-usage bias, resulting from synonymous substitutions at all third codon positions and first codon positions of leucine and arginine, as the main cause for the conflicting phylogenetic signals. We argue that the protein-coding gene data analyses are likely misleading due to artifacts induced by convergent composition biases at first codon positions of leucine and arginine and at all third codon positions. Our analyses corroborate previous studies based on gene sequence analysis that suggest Cyanobacteria evolved by the early paraphyletic splitting of Gloeobacter and a specific Synechococcus strain (JA33Ab), with all other remaining cyanobacterial groups, including both unicellular and filamentous species, forming the sister-group to the Archaeplastida lineage. In addition, our analyses using better-fitting models suggest (but without statistically strong support) an early divergence of Glaucophyta within Archaeplastida, with the Rhodophyta (red algae), and Viridiplantae (green algae and land plants) forming a separate lineage.

BioConvert: a comprehensive format converter for life sciences.

Bioinformatics is a field known for the numerous standards and formats that have been developed over the years. This plethora of formats, sometimes complementary, and often redundant, poses many challenges to bioinformatics data analysts. They constantly need to find the best tool to convert their data into the suitable format, which is often a complex, technical and time consuming task. Moreover, these small yet important tasks are often difficult to make reproducible. To overcome these difficulties, we initiated BioConvert, a collaborative project to facilitate the conversion of life science data from one format to another. BioConvert aggregates existing software within a single framework and complemented them with original code when needed. It provides a common interface to make the user experience more streamlined instead of having to learn tens of them. Currently, BioConvert supports about 50 formats and 100 direct conversions in areas such as alignment, sequencing, phylogeny, and variant calling. In addition to being useful for end-users, BioConvert can also be utilized by developers as a universal benchmarking framework for evaluating and comparing numerous conversion tools. Additionally, we provide a web server implementing an online user-friendly interface to BioConvert, hence allowing direct use for the community.

piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development.

Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.

Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis.

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.

Germline inherited small RNAs facilitate the clearance of untranslated maternal mRNAs in C. elegans embryos.

Inheritance and clearance of maternal mRNAs are two of the most critical events required for animal early embryonic development. However, the mechanisms regulating this process are still largely unknown. Here, we show that together with maternal mRNAs, C. elegans embryos inherit a complementary pool of small non-coding RNAs that facilitate the cleavage and removal of hundreds of maternal mRNAs. These antisense small RNAs are loaded into the maternal catalytically-active Argonaute CSR-1 and cleave complementary mRNAs no longer engaged in translation in somatic blastomeres. Induced depletion of CSR-1 specifically during embryonic development leads to embryonic lethality in a slicer-dependent manner and impairs the degradation of CSR-1 embryonic mRNA targets. Given the conservation of Argonaute catalytic activity, we propose that a similar mechanism operates to clear maternal mRNAs during the maternal-to-zygotic transition across species.