Recent developments in epigenetics suggest the widespread use of small-RNA pathways as a genomic immune system allowing eukaryotes to control molecular parasites such as transposable elements (TEs). Small RNAs have also been shown to mediate non-Mendelian inheritance in divergent species, resurrecting the Lamarckian idea of inheritance of acquired traits. To understand the basic biological logic that may underlie the connection between these themes, and identify fundamental aspects despite the confounding diversity of mechanisms uncovered in different phyla, we propose to address this issue from a phylogenetically unique angle. The ciliate Paramecium tetraurelia – at an equal evolutionary distance from plants and animals – was one of the first organisms in which transgenerational epigenetic inheritance was clearly demonstrated, and recent molecular studies have shown that many cases can be explained by the role of a meiosis-specific class of small RNAs, the scnRNAs, in epigenetic regulation of the genome rearrangements that occur during the development of the somatic macronucleus (MAC) from the germline micronucleus (MIC), in each sexual generation. Rearrangements include the elimination of all TEs as well as the precise excision of ~45.000 single-copy Internal Eliminated Sequences (IESs), which are believed to be degenerate remnants of ancient TE insertions. MIC centromeres may also be eliminated since they are not active in the MAC, which divides by a non-mitotic mechanism. During meiosis, scnRNAs are produced from the entire MIC genome and mediate a genome-wide comparison of germline and somatic sequences, allowing the zygotic MAC to eliminate any germline sequence not present in the maternal MAC. The system has probably evolved as an efficient mechanism to detect any new TE insertion in the germline, and has further been co-opted to ensure the non-Mendelian inheritance of essential phenotypic polymorphisms, such as mating types. But the mechanism should also in principle make any new insertion potentially lethal if it is introduced by conjugation into a naive cell - an effect that is reminiscent of hybrid dysgenesis in Drosophila, the devastating effects of TEs introduced by the male gamete when the mother cannot produce homologous piRNAs to repress transposition. By comparing the IES content of paralogous gene pairs of different ages derived from 3 successive whole-genome duplications, we recently obtained evidence that a substantial fraction of IESs have been acquired since the last duplication. This is the time when the Paramecium ancestor underwent numerous speciation events, resulting in a group of 15 sibling species that are morphologically indistinguishable but sexually incompatible. This raises the possibility that the scnRNA-based mechanism for recognition and elimination of TEs, which decreases the burden on host fitness and allows them to persist in the genome and degenerate into single-copy IESs, has been a major force driving speciation. The main objectives of this project are to reconstruct the evolutionary history of IESs and other MIC specific sequences such as TEs and centromeres, and to experimentally test the hypothesis that polymorphisms in these elements result in sexual incompatibility. We propose to (i) sequence and assemble the entire MIC genome of P. tetraurelia, since very little is currently known about the diversity and copy number of TEs; (ii) explore further the scnRNA pathway mechanism, by testing the role of known protein factors in the recognition of the genome-wide set of ~45,000 IESs, by identifying new ones, and by deep-sequencing scnRNA populations during sexual events to describe their dynamics; (iii) identify IESs and centromeres in different strains and species to study their evolution; and (iv) experimentally determine the effects of IES polymorphisms (presence/absence, divergence of sequences) on genome rearrangements in sexual progeny of interstrain or interspecies crosses.

Detyrosination is a post-translational modification associated with stable microtubules, which consists in the removal of the last amino acid from the C-terminus of alpha-tubulin. Up to now, two families of detyrosinases have been identified in mammalian cells. Nevertheless, these families are not conserved in Drosophila where detyrosination takes place specifically in the male germline. Through a genetic screen we recently identified a third class of detyrosinase acting during fly spermatogenesis. The first part of this project aims at characterizing the function of this enzyme and the role of detyrosination in the male germline. Strikingly, we provide strong evidence that a female-specific isotype of aplha-tubulin, which carries a terminal phenylalanine, is similarly processed in the female germline, by another enzyme. Based on our recent discovery of the new detyrosinase TMCP1 in mammals (Nicot et al. Sci Adv 2023) and of a new detyrosinase in Drosophila, we propose in a second part a strategy to identify this unknown “dephenylalaninase” and analyze its function. This project will therefore lead to the characterization of two new families of aplha-tubulin carboxy-peptidases and paves the way for the identification of their putative functional homologs in mammals.

Due to their capacity to jump from one site to another, transposable elements (TEs) are a significant threat to genome integrity. To limit TE mobilization, most eukaryotes have evolved small RNAs (sRNAs) to silence TE activity via homology-dependent mechanisms. sRNAs guide PIWI proteins to which they bind to homologous TE sequences, recruit histone-modifying enzymes, and repress the transcriptional activity of TEs. However, how small RNA-PIWI complexes recruit downstream effectors remains elusive. We will address this important question using the sRNA-guided genome elimination paradigm in ciliates. In the ciliate Paramecium, massive and reproducible elimination of TEs occurs during the development of the somatic genome from the germline genome at each sexual cycle. The specific recognition of TEs involves sRNAs which direct heterochromatin formation and subsequent TE elimination. sRNAs are produced from the entire germline genome, from TEs and non-TE sequences, by a developmental-specific RNAi interference pathway. Non-TEs sRNAs, which represent a large fraction of the sRNA population, are degraded, while sRNAs corresponding to TEs will remain and recruit Polycomb Repressive Complex 2 (PRC2) and trigger TE elimination. How non-TEs sRNAs are massively degraded and TE sRNAs are selected is currently unknown. In this proposal, we aim at uncovering how sRNAs are selectively degraded in Paramecium, an exquisite model because of the abundance and precise developmental timing of sRNA degradation. Our preliminary data identified new factors required for the process. We will use multidisciplinary, cutting-edge approaches, combining functional genomics and proteomics, to decipher the mode of action of these factors and the pathways involved.

Natural mucin gels are ubiquitous in animals. They display remarkable physicochemical properties and play important roles such as engulfment of pathogens, cancer progression and adhesion. Yet our understanding of these biomaterials is currently limited because we lack a simple genetic system to manipulate and produce them. This project aims to use a new tractable model that we recently developed, the glue produced by Drosophila larvae, to investigate how mucins and glue secretion have evolved and adapted to various environments. We will characterize the diversity of glue adhesive properties of several Drosophila species in various conditions. We will also use available RNAi lines to uncover the role of the various glue components in the adhesive properties. Furthermore, we will identify the glue genes conferring very strong adhesion in certain Drosophila species using RNAseq and functional validation in D. melanogaster. Finally, we will study how glue secretion has changed from one gland to another during the evolution of Phoridae flies. Our work should provide original data on the molecular mechanisms underlying species adaptation and bioadhesion.

Cell division is crucial for the development of complex organisms, for the homeostasis of tissues, and for the reproductive capacity of individuals. While most somatic cells are diploid and proliferate through mitosis, multiplication of sexually reproducing species relies on haploid gametes that are generated through a specialized cell division process called meiosis. To achieve this reduction in ploidy, two rounds of chromosome segregation follow a single phase of genome replication. Inaccuracy in this process leads to gametes that carry an incorrect number of chromosomes and to aneuploid embryos after fertilization. In their vast majority, these are non-viable and lead to spontaneous abortion: defective meiotic division is therefore a major obstacle in achieving reproduction. However, the key principles that drive this process are still poorly understood, one main reason being the diversity of the molecular scenarios that have been adopted across evolution to regulate oocyte chromosome segregation. Unlike any other type of cell division, reductional meiosis I leads to the segregation of chromosomes and not sister chromatids. This requires sister kinetochores, the macromolecular assemblies that link each chromosome to spindle microtubules, to function as a single unit. How this meiotic adaptation, which is essential for successful gamete production and reproduction, is achieved remains unclear. To dissect the key components of successful oocyte meiotic chromosome segregation, we propose to carry out a multi-disciplinary approach, combining two powerful model organisms, the nematode Caenorhabditis elegans and mice, with the use of cutting edge high-resolution live and electron microscopy methods. C. elegans and mice both produce haploid oocytes, but display drastically different chromosomal and kinetochore architectures. Our common project should therefore also provide an evolutionary perspective on oocyte meiosis. For this, we will: (AIM1) analyse meiotic kinetochore assembly and composition by immunostaining, live cell imaging and super resolution microscopy. Microtubule attachment sites will be identified by 3D-electron tomography, and the site and amplitude of forces applied to meiotic kinetochores in meiosis I and II will be determined with FRET-based tension sensors. (AIM 2) we will then assess whether a structure physically fusing sister kinetochores can be detected by performing Serial Block Face-Scanning Electron Microscopy (SBF-SEM) on high pressure frozen oocytes to reconstruct the whole meiosis I kinetochore in 3D. We will ask whether co-orientation and fusion of sister kinetochores depend on centromere-localized cohesion, on recombination, and/or on a protein that was previously proposed to fuse kinetochores together in mouse oocyte meiosis I. (AIM 3) finally, we will determine whether the meiotic segregation pattern is determined intrinsically by the chromosomes and/or by the cell cycle stage of the oocyte. Functional data will be obtained by employing a large panel of mouse and C. elegans genetic tools that are already available in the two partner groups, and sophisticated multimodal and latest state of the art. The combined expertise of the two partners and the large panel of tools already generated by both teams should allow successful implementation of the project to generate results that will have important implications for our understanding of meiotic cell division in oocytes.