Biology subjects

Betermier, M.

Publications and source records attributed to Betermier, M..

7 recordsLinked to original sources

bioRxiv2025

The tiny germline chromosomes of Paramecium aurelia have anexceptionally high recombination rate and are capped by a new class of Helitrons

BackgroundParamecia belong to the ciliate phylum of unicellular eukaryotes characterized by nuclear dimorphism. A diploid germline micronucleus (MIC) transmits genetic information across sexual generations. A polyploid transcriptionally active somatic macronucleus (MAC) develops at each sexual generation from a copy of the MIC through programmed DNA elimination (PDE) of > 30% of germline DNA. PDE requires the domesticated PiggyMac (Pgm) transposase. Assembly of Paramecium germline genomes has presented an enormous challenge owing to the difficulty of MIC isolation. ResultsWe report chromosome-scale short-read MIC assemblies for 7 species from the P. aurelia species complex. We discovered a novel clade of Helitrons, with 9-10 kb transposase ORFs under purifying selection, that have remained active in all P. aurelia lineages. A long-read assembly for P. tetraurelia together with a genetic linkage map provided a nearly telomere-to-telomere assembly. Conclusions.The genome consists of tiny (300 kb - 1.2 Mb) and numerous ([~]160) germline chromosomes with the highest recombination rate ever reported for a eukaryote (420 cM/Mb). The ends of the chromosomes consist of Helitrons inserted in telomeric C4A2 repeats, forming a distinct genomic compartment that is eliminated very early during MAC development in a Pgm-independent manner.

genomics↗

bioRxiv2025

A developmental condensin I complex assists the Paramecium PiggyMac domesticated transposase during programmed DNA elimination

Prokaryotes and eukaryotes use diverse strategies to cope with invading mobile genetic elements, including programmed DNA elimination (PDE). In the ciliate Paramecium, elimination of transposable elements and their relics requires the PiggyMac (Pgm) endonuclease and its five PgmL partners, yet how this machinery is targeted to cleavage sites remains unclear. Here, we identified condensin I subunits in the proximity proteomes of Pgm and PgmL4. We show that they belong to a condensin complex that is essential for PDE and localizes to developing somatic nuclei. Depleting the development-specific subunits of this complex blocks DNA elimination, phenocopying a Pgm depletion. Developmental condensin is required for the correct nuclear localization of Pgm and some of the PgmLs. Moreover, Pgm and these PgmLs co-immunoprecipitate with condensin I. Our findings uncover functional and physical interactions between a eukaryotic DNA cleavage machinery and a specialized condensin complex that is critical for PDE in a non-dividing nucleus.

molecular biology↗

bioRxiv2024

A development-specific POLX essential for programmed genome rearrangement in Paramecium tetraurelia

During the sexual cycle, programmed genome rearrangement (PGR) in Paramecium tetraurelia involves the non-homologous end joining (NHEJ) DNA repair pathway to eliminate specific germinal Internal Eliminated Sequences (IESs) from the newly developing somatic nucleus. In addition to the core NHEJ factors Ku70/80 and Xrcc4/Lig4, additional enzymes are required to process the 4-base 5-protruding ends generated following DNA cleavage at IES boundaries, prior to their ligation. Here, we report that PolX (a,b,c,d), four P. tetraurelia distant orthologs of the human Pol{lambda} DNA polymerase, are involved in repair of IES excision junctions. During rearrangements, PolX-depleted cells accumulate genome-wide errors, such as unrepaired double-strand breaks, 1-nucleotide deletions and IES retention. Although all PolX paralogs can process DNA ends, two of them (PolXa&b) are induced during PGR and have acquired tight nuclear anchoring properties through their N-terminal region, which contains a predicted BRCT domain. Finally, we show that PolXa accumulates in nuclear foci together with other NHEJ proteins and the Dicer-like enzyme Dcl5, which is involved in the biogenesis of IES-specific small RNAs. We propose that these "DNA repair foci" correspond to the sites where IES concatemers, a by-product of IES excision, are ligated together to produce the precursors of iesRNAs.

molecular biology↗

bioRxiv2023

The nuclear PIWI-interacting protein Gtsf1 controls the selective degradation of small RNAs in Paramecium

Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gtsf1 is exclusively localized in the maternal macronucleus and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.

genetics↗

bioRxiv2023

Inter-generational nuclear crosstalk links the control of gene expression to programmed genome rearrangements during the Paramecium sexual cycle

Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangements (PGR) remove transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR up-deregulates key PGR genes, together with [~]600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.

genomics↗

bioRxiv2022

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics

With its nuclear dualism, the ciliate Paramecium constitutes an original model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MIC) and a polyploid somatic macronucleus (MAC) that develops from the MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of [~]45,000 TE-derived Internal Eliminated Sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by non-coding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with next-generation sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), while TEs are eliminated at a later stage. We show that time, rather than replication, controls the progression of DNA elimination. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision, by acquiring stronger sequence determinants and escaping epigenetic control.

genomics↗

bioRxiv2021

GC content but not nucleosome positioning directly contributes to intron-splicing efficiency in Paramecium

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of [~]151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.

genomics↗