Inter-generational nuclear crosstalk links the control of gene expression to programmed genome rearrangement 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 rearrangement (PGR) removes 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 upregulates key known 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.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Chromosome-scale assembly of the yellow mealworm genome.

Background: The yellow mealworm beetle, Tenebrio molitor, is a promising alternative protein source for animal and human nutrition and its farming involves relatively low environmental costs. For these reasons, its industrial scale production started this century. However, to optimize and breed sustainable new T. molitor lines, the access to its genome remains essential. Methods: By combining Oxford Nanopore and Illumina Hi-C data, we constructed a high-quality chromosome-scale assembly of T. molitor. Then, we combined RNA-seq data and available coleoptera proteomes for gene prediction with GMOVE. Results: We produced a high-quality genome with a N50 = 21.9Mb with a completeness of 99.5% and predicted 21,435 genes with a median size of 1,780 bp. Gene orthology between T. molitor and Tribolium castaneum showed a highly conserved synteny between the two coleoptera and paralogs search revealed an expansion of histones in the T. molitor genome. Conclusions: The present genome will greatly help fundamental and applied research such as genetic breeding and will contribute to the sustainable production of the yellow mealworm.

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the 'TruSeq (TS)' protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol 'TS5', which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

The Polycomb protein Ezl1 mediates H3K9 and H3K27 methylation to repress transposable elements in Paramecium.

In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.