Biology subjects

Roignant, J.-Y.

Publications and source records attributed to Roignant, J.-Y..

14 recordsLinked to original sources

bioRxiv2026

Alternative splicing and gene expression repurpose ancestral genes through distinct evolutionary routes

Specialized tissues often evolve by repurposing ancestral genes. Tissue-specific alternative splicing (TS-AS) and tissue-specific gene expression (TS-GE) have been proposed as complementary mechanisms contributing to tissue specialization, but whether they provide broadly interchangeable solutions or instead follow distinct, constrained evolutionary routes remains unknown. To address this question, here we compare TS-AS and TS-GE across eight homologous tissues, 20 bilaterian species and 7,178 ancestral gene families spanning 700 million years. The two mechanisms were similarly prevalent and, together, they affected nearly three-quarters of ancestral gene families across our dataset. However, they targeted largely non-overlapping gene sets within species, with contrasting architectures and functions. TS-AS usually acts on longer, exon-rich genes and modulates intracellular machinery shared across tissues, whereas TS-GE preferentially evolves in duplicated genes and impacts tissue-defining extracellular and nuclear processes. Unexpectedly, although TS-GE was globally more conserved, neural-specific splicing gains outnumbered expression gains at the origins of vertebrates and insects, revealing a prominent contribution of TS-AS to early nervous system transcriptome diversification. Exon-level orthology reconstructions further uncovered extensive recurrent evolution of neural-specific exons in the same ancestral genes across the phylogeny. Importantly, deletion of two such microexons in Drosophila negatively impacted overall fitness and led to neurological-associated phenotypes such as reduced climbing or hyperactivity. Altogether, our findings reveal distinct but complementary evolutionary routes by which gene expression and alternative splicing repurpose ancestral genes, and highlight recurrent neural exon evolution as a functionally important contributor to nervous systems.

evolutionary biology↗

bioRxiv2025

Pus7 mutation links tRNA dysregulation to aggressive behavior through activation of the integrated stress response and glycolytic reprogramming

Pseudouridine ({Psi}) is a prevalent RNA modification found in multiple RNA species. It is deposited by {Psi} synthases (Pus) and it can stabilize RNA structures. Patients carrying alterations in the Pus7 gene suffer from developmental delay, intellectual disability, microcephaly, hyperactivity and increased aggression levels. Here we show that the Pus7 mutation in human patient cells and in a Drosophila model is associated with a specific decrease of tRNA:Aspartate (tRNA-Asp) levels, which leads to slow decoding at Aspartate codons. This in turn activates the integrated stress response and induces a metabolic shift towards increased glycolysis and reduced mitochondrial respiration. Elevating tRNA-Asp expression, inhibiting the integrated stress response or dampening the glycolytic pathway is sufficient to rescue the aggressiveness phenotype, demonstrating the involvement of the tRNA-Asp-ISR-glycolysis axis in this behavior. Together our data provide new insights into the molecular defects associated with the loss of Pus7 and suggest potential new avenues for therapeutic treatment.

genetics↗

bioRxiv2025

Tissue specific function of the Drosophila 7SK snRNP in controlling motoneuron synaptic growth

The 7SK snRNP is a ribonucleoprotein complex comprising the non-coding RNA 7SK and the associated proteins MePCE, LARP7, and HEXIM. It regulates transcription in higher eukaryotes by sequestering the positive transcription elongation factor (P-TEFb), preventing premature entry of RNA Polymerase II in elongation. Loss of LARP7 in humans causes the Alazami syndrome, marked by restricted growth, impaired movement, and intellectual disability, though the underlying mechanisms remain unclear. In this study, we show that loss of Larp7 or 7SK RNA in Drosophila is viable but impairs locomotion and reduces axonal growth at neuromuscular junctions. Larp7 is enriched in specific motoneurons, where it functions autonomously to promote axogenesis. Reducing P-TEFb abundance partially rescues the locomotion and axonal growth defects, indicating that the 7SK complex mediates this function via transcriptional regulation. Transcriptomic analysis of mutant motoneurons revealed that the 7SK complex primarily regulates long genes with high GC content at their promoters. These findings provide new insights into the tissue-specific roles of the 7SK snRNP in transcription and organismal function.

developmental biology↗

bioRxiv2023

Enhanced detection of RNA modifications and mappability with high-accuracy nanopore RNA basecalling models

In recent years, nanopore direct RNA sequencing (DRS) has established itself as a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling errors in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low errors compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the error signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine ({Psi}) and N1-methylpseudouridine (m1{Psi}) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability and basecalling accuracy in nanopore DRS datasets.

genomics↗

bioRxiv2023

Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

molecular biology↗

bioRxiv2021

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

Neurons draw on alternative splicing for their increased transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. We found that in Drosophila, this splicing program is restricted to neurons by the post-transcriptional processing of the enhancer of microexons (eMIC) domain in Srrm234 by Elav and Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell-type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. Remarkably, we found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient post-transcriptional programs can evolve independently in different phyla to impact distinct cellular modules while maintaining cell-type specificity.

molecular biology↗

bioRxiv2021

An exon junction complex-independent function of Barentsz in neuromuscular synapse growth

The exon junction complex controls the translation, degradation and localization of spliced mRNAs, and three of its four core subunits also play a role in splicing. Here we show that the fourth subunit, Barentsz, has distinct biological functions within and separate from the exon junction complex in neuromuscular development. Barentsz controls the distribution of mitochondria in larval muscles, a function that also depends on other subunits of the exon junction complex and that is not rescued by a transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We found that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.

developmental biology↗

bioRxiv2020

The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein

N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. The modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6A alters fly behavior albeit the underlying mechanism and the role of m6A during nervous system development have remained elusive. Here we find that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system being required for limiting axonal growth. Mechanistically, we show that Ythdf directly interacts with Fragile X mental retardation protein to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection.

molecular biology↗

bioRxiv2020

m6A RNA methylation regulates promoter proximal pausing of RNA Polymerase II

RNA Polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing. The m6A methyltransferase complex (MTC), with the nuclear reader Ythdc1, are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body, and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

molecular biology↗

bioRxiv2019

tRNA 2-O-methylation modulates small RNA silencing and life span in Drosophila.

2-O-methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two novel Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1, respectively. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while subsequently, CG5220 methylates position C32 in the same tRNAs and targets also additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as life span reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalysing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

molecular biology↗

bioRxiv2019

The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A)-translation

Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 interacts with the cytoplasmic poly(A)-binding protein (PABP) and is positioned upstream of poly(A) tails in mRNAs. Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10, as well as additional translational regulators as main ubiquitylation substrates of MKRN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.

molecular biology↗

bioRxiv2018

Makorin1 controls embryonic patterning by alleviating Bruno-mediated repression of oskar translation

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning and germ cell specification. Here, we show that Mkrn1 is required for translational activation of oskar, whose product is critical for axis specification and germ plasm assembly. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds osk 3 UTR in a region adjacent to A-rich sequences. This binding site also overlaps with Bruno (Bru) responsive elements (BREs), which regulate osk translation. We observe increased association of the translational repressor Bru with osk mRNA upon depletion of Mkrn1, implying that the two proteins compete for osk binding. Consistently, reducing Bru dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation via displacing Bru from its 3 UTR.

developmental biology↗

bioRxiv2018

The exon junction complex regulates the release and phosphorylation of paused RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) plays a critical and conserved role in this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.

molecular biology↗

bioRxiv2017

Comprehensive Characterization Of The Complex Lola Locus Reveals A Novel Role In The Octopaminergic Pathway Via Tyramine Beta-Hydroxylase Activation

longitudinals lacking (lola) is among the most complex genes in Drosophila melanogaster, encoding up to twenty protein isoforms and acting as a key transcription factor in axonal pathfinding and neural reprogramming. Most of previous studies employed loss-of-function alleles disrupting common exons of lola, making it difficult to delineate its functions. To address this issue we have generated specific mutations in each isoform using the CRISPR/Cas9 system. Our targeted screen allows us to revisit the previously demonstrated roles for few isoforms and to demonstrate a specific function for one variant in axon guidance via activation of the microtubule-associated factor Futsch. Importantly, we also reveal a critical role for a second variant in preventing neurodegeneration via the control of the octopaminergic pathway. This variant is expressed almost exclusively in the octopaminergic cells and is involved in the transcriptional activation of a key enzyme of the pathway. Thus, our comprehensive study greatly expands the functional repertoire of Lola functions, and adds novel insights into the transcriptional regulatory control of neurotransmitter expression in vivo.

neuroscience↗