Biology subjects

Barbier, J.

Publications and source records attributed to Barbier, J..

11 recordsLinked to original sources

bioRxiv2025

NRDE2 Interacts with an Early Transcription Elongation Complex and Widely Impacts Gene Expression

NRDE2 is a highly conserved protein implicated in post-transcriptional gene silencing in Schizosaccharomyces pombe and Caenorhabditis elegans and has been shown to modulate splicing in mammals. To explore whether NRDE2 participates in additional processes in human cells, we performed tandem affinity purification followed by proteomic analysis of NRDE2 from nuclear extracts of HEK293T and HeLa cells. Our analysis confirmed the interaction of NRDE2 with its well-characterized partner, the MTR4 helicase (MTREX), as well as with multiple splicing factors. Notably, we also identified interactions with chromatin-associated proteins involved in transcription, including the Polymerase-Associated Factor 1 (PAF1) complex and elongating forms of RNA polymerase II (RNAPII). To further investigate NRDE2 function, we conducted RNA-seq following its transient depletion. Differential expression analysis revealed that loss of NRDE2 alters the expression of thousands of genes. Consistent with earlier reports, we observed splicing defects, particularly intron retention; however, our results indicate that the impact of NRDE2 on intron retention is more extensive than previously recognized. Moreover, intron retention was frequently associated with reduced mRNA expression. Together, these findings suggest that NRDE2 associates with both the transcriptional and splicing machineries and plays a broader role in RNA processing than previously appreciated.

molecular biology↗

bioRxiv2023

Decoding Nucleosome-Depleted Regions: Insights from Epigenetic Marks, Nucleosome Size, and Thermodynamic Modelling

Elucidating the global and local rules that govern genome-wide nucleosome organisation and chromatin architecture remains a critical challenge. Thermodynamic modelling based on DNA elastic properties predicts the presence of sequence-encoded nucleosome-inhibiting energy barriers (NIEBs) along vertebrate genomes. They delineate in vivo nucleosome-depleted regions (NDRs) flanked by 2-3 well positioned nucleosomes. Here, we compared mouse NIEBs to NDRs observed at CTCF binding sites and active TSSs to reveal specific chromatin organizations. We uncover in MNase-seq chromatin profiles the presence of particles of subnucleosomal length specifically positioned at the border of NIEBs with an enrichment of H3.3 and its modification H3.3 S31Ph, whereas the positioning of nucleosomes bearing H3K27ac appears insensitive to NIEBs. Surprisingly, post-translational modifications affect the size distribution of nucleosomes as seen by MNase digestion and so likely their breathing capability. We implemented an extension of our thermodynamic model allowing for variable particle size and suggest that subnucleomes at NIEB borders would result from the recruitment of chromatin remodellers at NIEBs. Our findings provide new insights into the mechanisms by which the DNA sequence and epigenetic marks shape the nucleosome positioning and breathing.

genomics↗

bioRxiv2023

Selective inhibition of OSBP blocks retrograde trafficking by inducing partial Golgi degradation

Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective small molecule modulators can be challenging due to structural similarities in the sterol binding domains. We report the discovery of highly potent and selective inhibitors of oxysterol binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol binding proteins by targeted degradation, led to a significant reduction in Golgi-associated proteins. The degradation was found to occur at lysosomes, concomitant with changes in general protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target any other sterol transporters tested, we advocate their use as chemical tools to study OSBP function and therapeutic relevance.

cell biology↗

bioRxiv2022

Plasma cell maintenance and antibody secretion are under the control of Sec22b-mediated regulation of organelle dynamics

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the SNARE Sec22b as a unique and critical regulator of plasma cell maintenance and function. In absence of Sec22b, plasma cells were barely detectable and serum antibody titres were dramatically reduced. Accordingly, Sec22b deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b is indispensable for efficient antibody secretion but also for plasma cell fitness through the regulation of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil a critical role for Sec22b-mediated regulation of plasma cell biology through the control of organelle dynamics.

immunology↗

bioRxiv2021

NF90 Interacts with Components of RISC and Modulates Association of Ago2 with mRNA

Nuclear Factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3 UTRs, NF90 association was increased upon loss of MOV10 and vice versa, suggesting that the two proteins may compete for the binding of common target mRNAs. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. These findings suggest a role for NF90 in the regulation of RISC-mediated silencing which stabilizes target mRNAs, such as VEGF, during cancer-induced hypoxia.

molecular biology↗

bioRxiv2021

A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

microbiology↗

bioRxiv2021

Bone innervation and vascularization regulated by osteoclasts contribute to refractive pain-related behavior in the collagen antibody-induced arthritis model

ObjectiveRheumatoid arthritis is often characterized by eroded joints and chronic pain that outlasts disease activity. Whilst several reports show strong associations between bone resorption and nociception, the underlying mechanisms remain to be unraveled. Here, we used the collagen antibody-induced arthritis (CAIA) model to examine the contribution of osteoclasts in pain regulation. The antinociceptive effects of osteoclasts inhibitors and their mechanisms of actions involving bone vascularization and innervation were also explored. MethodsBALB/c female mice were subjected to CAIA by intravenous injection of a collagen type-II antibody cocktail, followed by intraperitoneal injection of lipopolysaccharide. Degree of arthritis, bone resorption, mechanical hypersensitivity, vascularization and innervation in the ankle joint were assessed. Animals were treated with osteoclast inhibitors, zoledronate and cathepsin K inhibitor (T06), and netrin-1 neutralizing antibody. Potential pronociceptive factors were examined in primary osteoclast cultures. ResultsCAIA induced local bone loss in the calcaneus with ongoing increased osteoclast activity during the inflammatory phase of the model, but not after inflammation has resolved. Mechanical hypersensitivity was reversed by zoledronate in late but not inflammatory phase CAIA. This effect was coupled to the ability of osteoclasts to modulate bone vascularization and innervation, which was inhibited by osteoclast inhibitors. CAIA-induced hypersensitivity in the late phase was also reversed by anti-netrin-1 antibody. ConclusionOsteoclasts induce pain-like behavior in the CAIA model independent of inflammation via effects on bone vascularization and innervation. Key messagesWhat is already known about this subject? O_LIPain and residual signs of erosive lesions are frequently present in rheumatoid arthritis (RA) patients with good disease control C_LIO_LIOsteoclasts can induce nociceptive signaling but the exact mechanism with respect to RA-induced pain is not clear C_LI What does this study add? O_LIThe pronociceptive actions of osteoclasts extend beyond flares of joint inflammation and erosive activity by increasing bone innervation, bone vascularization and netrin-1 release C_LIO_LIOsteoclast inhibitors and neutralizing netrin-1 antibodies reverse refractive pain-related behaviors in the collagen antibody-induced arthritis model C_LI How might this impact on clinical practice or future developments? O_LIThis study provides insights to the potential of osteoclast inhibition as a therapeutic strategy for persistent pain in RA C_LI

neuroscience↗

bioRxiv2021

Lysophophatidyl-choline 16:0 mediates persistent joint pain through Acid-Sensing Ion Channel 3: preclinical and clinical evidences

Rheumatic diseases are often associated to debilitating chronic joint pain, which remains difficult to treat and requires new therapeutic strategies. Here, we describe increased content of lysophosphatidyl-choline (LPC) 16:0 in the knee synovial fluids of two independent cohorts of patients with painful joint diseases. If LPC16:0 levels correlated with pain in patients with osteoarthritis (OA), they do not appear to be the hallmark of a particular joint disease. We found that intra-articular injections of LPC16:0 in mouse produce chronic pain and anxiety-like behaviors in both males and females with no apparent inflammation, peripheral nerve sprouting and damage, nor bone alterations. LPC16:0-induced persistent pain state is dependent on peripheral Acid-Sensing Ion Channel 3 (ASIC3), ultimately leading to central sensitization. LPC16:0 and ASIC3 thus appear as key players of chronic joint pain with potential implications in OA and possibly across others rheumatic diseases.

neuroscience↗

bioRxiv2020

PAF1 facilitates RNA polymerase II ubiquitination by the Elongin A complex through phosphorylation by CDK12

The conserved Polymerase-Associated Factor 1 complex (PAF1C) regulates all stages of the RNA polymerase II (RNAPII) transcription cycle from the promoter to the 3 end formation site of mRNA encoding genes and has been linked to numerous transcription related processes. Here, we show that PAF1 interacts with Elongin A, a transcription elongation factor as well as a component of a cullin-RING ligase that targets stalled RNAPII for ubiquitination and proteasome-dependent degradation in response to DNA damage or other stresses. We show that, in absence of any induced stress, PAF1 physically interacts with the E3 ubiquitin ligase form of the Elongin A complex and facilitates ubiquitination of RNAPII. We demonstrate that this ubiquitination is dependent of the Ser2 phosphorylation of the RNAPII carboxy-terminal domain (CTD) by CDK12. Our findings highlight a novel unexpected role of PAF1-CDK12 in RNAPII transcription cycle, raising the possibility that the Elongin A ubiquitin ligase plays a role in normal transcription process, and suggest a transcription surveillance mechanism ready to degrade RNAPII if needed.

cell biology↗

bioRxiv2020

NF90 Modulates Processing of a Subset of Human Pri-miRNAs

MicroRNAs are predicted to regulate the expression of more than 60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The dsRNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of pri-miRNAs. Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA EMSA. NF90-bound and modulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly modulated. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

molecular biology↗

bioRxiv2018

Transiently depleting RNPS1 leads to perdurable changes in alternative splicing

While robust regulatory mechanisms are expected to control the production of splice variants that confer distinct functions, a low level of stochasticity may be tolerated. To investigate stringency of regulation, we followed changes in the splicing of 192 alternative cassette exons after growth of cancer-derived HCT116 cells and embryonic colonocytes. In both cell lines approximately 15% of alternative splicing events changed by more than 10 percentage points over a 42-day period. We then carried out a cycle of transient depletions targeting RNPS1, a splicing regulator implicated in genomic stability. For alternative splicing units not regulated by RNPS1, the level of splicing changes was similar to the stochastic value obtained after normal growth. However, the frequency of perdurable changes was at least twice that value for splicing events regulated by RNPS1. A swap allele assay performed on four RNPS1-responsive units that underwent splicing changes indicated the presence of mutations mediating this effect. Specifically, a T to C mutation in a RNPS1-responsive exon of ADARB1 confered exon skipping. Our results suggest that fluctuations in the level of a splicing regulator preferentially impact the integrity of genes encoding transcripts that are regulated by this splicing factor to produce perdurable changes in alternative splicing. We discuss the potential implication of this process in human evolution.

molecular biology↗