IMCBio International PhD Program

The ~30,000 nucleotide long SARS-CoV-2 RNA serves as mRNA for translation of the non-structural precursor proteins, as a template for synthesis of genomic and subgenomic RNAs, and as genomic RNA (gRNA) that must be specifically incorporated in the new viral particles. Incorporation of the SARS-CoV-2 gRNA is coupled with viral assembly, which takes place at the endoplasmic reticulum-Golgi intermediate complex (ERGIC). The N protein (nucleoprotein) plays a central role in this process by binding to yet unidentified packaging signals (PS) (1). In vitro, RNA binding to N causes liquid-liquid phase separation (LLPS), and this phenomenon might be involved in RNA packaging. Interestingly, in mouse hepatitis virus and closely related lineage A betacoronaviruses, which are the only coronaviruses for which the PS have been precisely identified, mutations in the PS not only affected RNA packaging and viral fitness, but mutant viruses also failed to overcome the innate immunity. Furthermore, targeting LLPS of SARS-CoV-2 N protein promotes innate antiviral immunity by elevating MAVS activity (2). Therefore, we hypothesize that binding of N to the PS might suppress the innate antiviral immune response.

We plan to (i) identify SARS-CoV-2 PS by analyzing RNA binding of wild-type N and of a collection of N mutants already available in the laboratory in vitro, (ii) analyze the effect of point mutations in PS on N binding in vitro and RNA packaging in virus-like particles (3), (iii) study the impact of these mutations on LLPS in vitro and in cells, and (iv) analyze the impact of the PS mutations on the MAVS activity.

1. Masters (2019) Virology 537, 198-217 (review)
2. Wang et al. (2021) Nat Cell Biol 23, 718-732

Syed et al. (2021) Science 374, 1626-1632

Key words: SARS-CoV-2, RNA packaging, N protein, Liquid-liquid phase separation, RNA sensing

Aptamers (i.e., single stranded nucleic acids adopting a 3D structure allowing them to specifically interact with a target molecule) and ribozymes (nucleic acids endowed with a catalytic function) represent a class of nucleic with growing interest in bioengineering, biomedical and even industrial fields. Lots of such natural and synthetic nucleic acids have been described over the past decades, all being usually functional in water-based environment.

In this project, we will explore a new generation of aptamers and ribozymes adapted to work in non-conventional solvents where water will be exchanged for ionic liquids. To reach this goal, mutant libraries will be functionally screened using droplet-based microfluidics as routinely done by the team. However, because of solvent exchange, a first step of the PhD work will consist in adjusting the design of the microfluidic devices to optimize the production and the manipulation of non-aqueous droplets. Then, these devices will be exploited to isolate optimized aptamers/ribozymes and identify most interesting molecules through sequence analysis (high-throughput sequencing in tandem with bioinformatics). Upon characterization, the newly isolated molecules will be transfer to partners where they will serve for various industrial applications.

This PhD project is part of a larger and more ambitious project that received the financial support from the European Innovation Council (EIC) and will involve 8 partners worldwide. We are looking for a highly motivated student with both excellent scientific and soft skills. We are more specifically looking for a candidate with strong background in molecular biology and an interest in technological development. Skills in programming (Python or R) will be a plus but are not mandatory. This PhD contract will be funded for 3 years starting from May 2023.

Keywords: synthetic nucleic acids, aptamers, ribozyme, microfluidics, droplets, innovation

Proteases are enzymes involved in plenty of biological and pathological processes, such as tissue degradation facilitating, for instance, invasion by pathogens or cancer cells dissemination. The goal of this project will be to set-up and perform the development of a new generation of aptamers (i.e., single stranded nucleic acids adopting a 3D structure allowing them to specifically interact with a target molecule) targeting secreted proteases. Several aptamers against proteases have already been described in the literature, but they were all made either of DNA or of RNA.

During this thesis, we will explore alternative backbone chemistries to produce aptamers featuring both by the structural plasticity of RNA and the extended halftime of DNA. To reach this goal, the student will use an integrated methodology combining in vitro selection (i.e., SELEX) and ultrahigh-throughout functional screening using droplet-based microfluidics (a unique expertise of the hosting laboratory) to enrich libraries in genes of interest. The use of Next Generation Sequencing in tandem with bioinformatics will then allow to identify hits that will be validated prior to being further characterized both structurally and functionally (e.g., affinity measurement, enzymatic characterization…). Strongly interdisciplinary, this project will combine chemical biology, molecular biology, microfluidics, and bioinformatics.

Keywords: In vitro evolution, aptamer, protease, microfluidics, high-throughput, innovation

Husser, C., Vuilleumier, S. and Ryckelynck, M.* (2022) FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride. Small. In press.

Bouhedda, F.; Fam, K.T.; Collot, M.; Autour, A.; Marzi, S.; Klymchenko, A.; Ryckelynck, M.* (2020) A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells. Nature Chemical Biology, 16, 69-76.

Autour, A.; Jeng, S.C.; Cawte, A.; Abdolahzadeh, A.; Galli, A.; Panchapakesan, S.S.; Rueda, D.*; Ryckelynck, M.*; Unrau, P.J.* (2018) Fluorogenic RNA Mango Aptamers for Imaging Small noncoding RNAs in Mammalian Cells. Nature Communications, 9, 656.

Cancer cells differ from normal cells displaying high level of proliferative capacity. That creates cancer cell vulnerabilities, which can be targeted therapeutically. One of the cellular machineries which are “hijacked” by cancer cells are the nuclear pore complexes (NPCs). NPCs constitute the sole communication gates between the nucleus and the cytoplasm and ensure cellular function and survival. Nucleoporins (Nups) are the building protein units of the NPCs and represent an “achilles heel” of cancer cells. Cancer cells increase their NPC number to adapt to proliferative demand and depletion of Nups required for NPC assembly can selectively kill cancer cells and reduce tumor growth, providing a strong proof of principle for targeting Nups in cancer disease. However, the molecular mechanisms on how rapid spatial assembly of the NPCs can be achieved in cancer cells and how exactly misregulation of Nups homeostasis can cause cancer disease remain largely unknown. Our published findings identified a novel mechanism on how spatial assembly and increased biogenesis of NPCs can be achieved in cancer cells. We plan to characterize the role of newly identified factors in Nups homeostasis in relevant cellular cancer models. Inhibition of these factors should selectively reduce viability of cancer cells. This research may create unprecedented therapeutic perspectives for cancer patients.

Keywords:

cancer, nuclear pore complexes, nucleoporins, nucleocytoplasmic transport, cancer-specific vulnerabilities

Gene expression requires ordered and efficient processing of messenger RNA (mRNA) molecules from their sites of synthesis in the nucleus to the cytoplasm, where they are translated into proteins. Yet, we know little about the cellular processes that orchestrate the flux of mRNA in a temporal and spatial manner. We recently discovered that the yeast NuA4 acetyl-transferase (KAT) complex, which acetylates histones to promote mRNA synthesis, is also required for the export of mRNA from the nucleus to the cytoplasm. This new export function requires acetylation of Nuclear Pore Complexes (NPCs), which are multiprotein complexes forming channels in the nuclear envelope. The role of NuA4 in mRNA export seems conserved in mammalian cells, because mouse embryonic stem (mES) cells lacking NuA4 KAT activity accumulate mRNA in nuclear phase-separated domains called nuclear speckles. The objective of this project is to determine how acetylation  regulates the transport of mRNAs from the nucleus to the cytoplasm in budding yeast and mES cells. Regulation of mRNA export may be particularly important during cell differentiation, which depends on rapid and sometimes extreme changes in gene expression. Thus, this project will reveal novel, key mechanisms regulating how the movement of mRNA through the cell controls cell identity and differentiation.

Key words: Nuclear pore complexes – acetylation – gene expression – mRNA synthesis and export – budding yeast – stem cells

The ribosomal DNA (rDNA) is a highly repetitive genomic region, which is essential to initiate ribosome biogenesis in the nucleolus. The repetitive architecture of rDNA and its high level of transcription promote genomic instability, resulting in frequent DNA damage, such as double-strand breaks (DSBs), which potentially lead to the excision of rDNA fragments forming extra-chromosomal rDNA circles (ERCs). In budding yeast, these ERCs have been described to be toxic for cellular homeostasis and to reduce longevity. Therefore, repairing efficiently DNA damage within the repetitive rDNA cluster is critical for cell survival.  It has been shown that DSBs within the rDNA locus are relocated from the nucleolus to nuclear pore complexes (NPC). These DSBs are subsequently repaired by homologous recombination. However, the detailed mechanism explaining how rDNA damage sites are transported to NPCs, repaired, and relocated to the nucleolus is still elusive. By combining two complementary powerful approaches, proximity proteomics and single-cell quantitative microscopy, we will identify the molecular players involved in rDNA damage response and characterize the mechanism of DSB site transport between the nucleolus and the nuclear envelope.

Keywords: DSB repair, rDNA, nucleolus, nuclear pore complex

It is now established that differentiated cells can change their identity. This fascinating event is called transdifferentiation (Td) when a differentiated cell is changed into another type of differentiated cell. We use as a model a natural Td event, which is remarkably 100% efficient, to understand the cellular and molecular mechanisms that allow a differentiated cell to change its identity (ID). What are the cellular steps between 2 identities? What type of transitions are involved: smooth, progressive transition from one ID or sharp transitions? How are these controlled? To answer these questions, we have purified and sequenced at the single cell level, both a cell that changes its identity, as well as its neighbour which looks identical but does not change its identity. Preliminary analyses of the scRNA-seq data obtained suggest a series of sharp and defined transitions. We propose here a thesis project to determine the core TFs and afferent Gene Regulatory Networks that control the different transition states during the process. A number of candidate transcription factors have already been identified that may drive different phases of this process. The project will consist of 1) Validating that the identified genes are necessary for Td and at what step. 2) Identifying their potential targets using our transcriptomic data. 3) Examining the hierarchical relationships that these genes may have with each other (e.g. transcriptional, subcellular localisation regulations). Our collaborator, the Molina lab, applied their recently established FateCompasss tool to predict not only which TFs are expressed but also which TFs are active over time. We will use these analyses to determine the important TF nodes, in order to propose a model of the molecular cascades involved in enabling a cell to be reprogrammed. The results will have important implications for the understanding of the basis of certain cancers, or to improve the safety and efficiency of our reprogramming strategies for regenerative therapies.

Key words: Cellular plasticity, transdifferentiation, SOX2, C. elegans, scRNA Seq, GRN

Relevant publications:

• Riva C., Gally C., Hajduskova M., Suman S.K., Ahier A. & Jarriault S. (2021) A natural transdifferentiation event involving mitosis is empowered by integrating signaling inputs with conserved plasticity factors. Cell Rep. 40(12):111365. doi: 10.1016/j.celrep.2022.111365
• Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans. Gómez-Saldivar G, Osuna-Luque J, Semple JI, Glauser DA*, Jarriault S*, Meister P*. Genetics. vol. 216 no. 4 931-945. (2020)   * co-last author
• Sequential histone-modifying activities determine the robustness of transdifferentiation. Zuryn S., Ahier A., Portoso M., Redhouse White E., Margueron R., Morin M.C. & Jarriault S. Science 345(6198):826-829.  (2014)  Cited « Recommended » by Faculty 1000.

Transcription controls the expression of genes, and thus the development and function of cells. Most eukaryotic genes are transcribed by RNA polymerase II (Pol II). Understanding the dynamics of this process in living cells is a fundamental question in biology. The candidate will participate in and further develop an ongoing project that addresses this question by measuring recruitment dynamics of endogenous Pol II and general transcription factors (GTFs) to an inducible gene array in live cells.

The candidate will monitor Pol II and GTFs recruitment dynamics to single visualisable genes using the antibody-based in vivo labeling approach. The candidate will use two different complementary approaches: i) set up a CRISP/Cas9-based technology to label a single endogenous locus in U2OS human osteocarcinoma cells. ii) monitor endogenous Pol II and GTF recruitment dynamics at single gene level using ANCHOR-based genome labeling. The recruitment of endogenous Pol II and GTFs to these endogenous engineered loci will be monitored by state-of-the-art imaging technics, including confocal and super resolution microscopy, upon transcription activation.

The results from these experiments will provide for the first-time description of pre-initiation complex assembly at different core promoters and gene regulation dynamics in live cells.

Keywords: Transcription regulation, RNA polymerase II, CRISPR/Cas9, genome editing, imaging, bioinformatics, promoter, live cells.

Relevant publications:

-Conic S., Desplancq D., Ferrand A., Fischer V., Heyer V., Reina San Martin B., Pontabry J., Oulad-Abdelghani M., Babu N.K., Wright G.D., Molina M., Weiss E. and Tora L. (2018) Imaging of native transcription factors and histone phosphorylation at high resolution in live cells. Journal of Cell Biology, 217(4):1537-1552. doi: 10.1083/jcb.201709153.

-Hadzhiev Y., Qureshi HK., Wheatley L., Cooper L., Jasiulewicz A., Nguyen H., Wragg J., Poovathumkadavil D., Conic S., Bajan S., Sik A., Hutvagner G., Tora L., Gambus A., Fossey J. and Müller F. (2018) A cell cycle-coordinated nuclear compartment for polymerase II transcription encompasses the earliest gene expression before global genome activation. Nature Communications, 10(1):691, doi: 10.1038/s41467-019-08487-5

- Wang F., El-Saafin F., Ye T., Stierle M., Negroni L., Durik M., Fischer V., Devys D., Vincent S.D. and Tora L (2021) Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription. Cell Death and Differentiation. 28(8):2385-2403. doi:10.1038/s41418-021-00759-2.

Cellular senescence is a form of irreversible cell cycle arrest induced by a variety of stimuli, including aging and chemotherapy. However, work from our lab has also shown how senescence, when present transiently, can have beneficial functions in embryonic development, plasticity and regeneration (e.g. Storer et al, Cell, 2013; Ristchka et al, Genes Dev., 2017). Our lab is interested in studying the cell biology of the senescence program in different settings: in physiological settings such as development and regeneration, and in pathological settings such as aging, disease and cancer.

We aim to recruit a Ph.D. student to investigate the effects of senescent cells on tumor progression and recurrence after therapy. It has been shown that senescent cells can boost tumor recurrence and metastasis, but the exact mechanisms remain unclear. We have shown previously that senescent cells promote cell plasticity and stemness that can be beneficial for regeneration (Ristchka et al, Genes Dev., 2017). Here, we aim to investigate if senescent cancer cells function similarly to promote tumor recurrence and metastasis. The project will be tailored to the students’ research interests, but will broadly involve the study of senescence in vivo using cell and molecular approaches, including mouse models, in vivo imaging, single cell sequencing and molecular biology.

Keywords: cellular senescence, cancer,  plasticity, development, regeneration, reprogramming, aging

Relevant publications:

• Durik, M., Sampaio Gonçalves, D., Spiegelhalter, C., Messaddeq, N., Keyes, W.M. Senescent cells deposit intracellular contents through adhesion-dependent fragmentation (2023) BioRxiv doi: https://doi.org/10.1101/2023.01.11.523642
• Ritschka, B., Knauer-Meyer, T., Sampaio-Conçalves, D., Mas, A., Plassat, J.L., Durik, M., Jacobs, H., Pedone, E., Di Vicino, U., Cosma, M.P. Keyes, W.M. (2020) The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice. Genes & Development, 34(7-8):489-494.
• Ritschka, B., Storer, M., Mas, A., Heinzmann, F., Ortells, M.C., Morton, J.P., Sansom, O.J., Zender, L. and Keyes, W.M. (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration Genes & Development, 31(2):172-183

Title: Role of Fam72a in antibody diversification.

During the course of immune responses B cells diversify their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR). These reactions establish highly specific and adapted humoral responses by increasing antibody affinity and by allowing the expression of a different antibody isotype with unique immunological functions. SHM diversifies the variable region Immunoglobulin heavy (IgH) and light (IgL) chain genes, producing families of related clones bearing mutated receptors that are positively selected on the basis of antigen binding affinity.

CSR diversifies the B cell repertoire by combining a single heavy chain variable region with a different constant region, switching the antibody isotype expressed (from IgM to IgG, IgE or IgA) while retaining the antigen specificity of the receptor. Both of these reactions are initiated by Activation Induced Cytidine Deaminase (AID), an enzyme that deaminates cytosines in DNA. AID-induced lesions are recognized by the DNA repair machinery and are processed in different ways to trigger mutations or double stranded DNA break intermediates during CSR.

At present in is not understood how AID-induced DNA damage results in error-prone DNA repair. We have recently conducted a genome-wide CRISPR/Cas9 knockout screen looking for genes involved in CSR. We have identified Fam72a as a key regulator in the balance between error-prone and error-free DNA repair (Rogier et al. Nature 2021). Here, we propose to further understand, at the molecular level, how Fam72a regulates the choice between error-free and error-prone DNA repair during antibody diversification. The project's goal is to generate individual knockouts in CH12 cells for a number of candidate genes which we suspect being involved and conduct initial mechanistic studies before generating conditional knockout mouse models to study SHM and CSR in vivo.

Keywords: Antibody diversification, DNA Repair, Class Switch recombination, CRISPR/Cas9.

Relevant publications:

• Rogier, M. et al. Fam72a enforces error-prone DNA repair during antibody diversification. Nature 600, 329, doi:10.1038/s41586- 021-04093-y (2021).
• Yilmaz, D. et al. Activation of homologous recombination in G1 preserves centromeric integrity. Nature, doi:10.1038/s41586-021- 04200-z (2021).
• Amoretti-Villa, R., Rogier, M., Robert, I., Heyer, V. & Reina-San-Martin, B. A novel regulatory region controls IgH locus transcription and switch recombination to a subset of isotypes. Cell Mol Immunol 16, 887-889 (2019).
• Thomas-Claudepierre, A.S., et al. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination. J Exp Med 213, 303-312 (2016).
• Thomas-Claudepierre, A.S., et al. The cohesin complex regulates immunoglobulin class switch recombination. J Exp Med 210,2495-2502 (2013).

Most eukaryotic mRNAs transcribed by polymerase II are post-transcriptionally modified with addition of a 5’ cap as well as a 3’ poly(A) tail, and splicing of introns. Some internal A residues are also methylated to form m6A contributing to the regulation of gene expression and beyond the control of development or pathologies. m6A modification has been reported to affect several processes including pre-mRNA splicing, translation and mRNA decay. Yet, the mechanism of action of m6A residues remain incompletely understood, in part because few functional m6A site have been identified. Indeed, if high throughput studies have mapped numerous m6A site, whether those have a biological function or are silent remains unclear.

In the yeast Saccharomyces cerevisiae, m6A formation occurs only during meiosis. Previous study from the host team identified residues bound by a “reader” protein that binds (some) m6A residues to carry out downstream biological function (Scutenaire et al., 2022). This study demonstrated that mRNA methylation controls meiotic recombination in yeast. Strikingly, preventing modification of a single A residue by mutation was sufficient to delay meiosis. Based on this observation, the impact of adenine methylation on expression of key mRNAs will be monitored to decipher mechanisms of action, including regulation of translation and mRNA decay. The project will involve in vivo analyses (characterization of mutants, analyses of reporter genes...), biochemical assays (activity tests, protein-RNA interaction studies...) as well as high-throughput analyses (transcriptomic, ribosome profiling). Results will contribute to understand the role of m6A at the interface between degradation of mRNAs and translation and to elucidate its physiological in yeast and beyond its links to genetic diseases and cancer.

Keywords: mRNA, m6A, regulation of gene expression, RNA decay, translation, diseases

Relevant publications:

- Scutenaire et al. The S. cerevisiae m6A-reader Pho92 promotes timely meiotic recombination by controlling key methylated transcripts. Nucleic Acids Res. 2022, gkac640. doi: 10.1093/nar/gkac640.

- Mauxion et al. The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions. Cell Rep. 2022, 111902. doi: 10.1016/j.celrep.2022.111902.

- Charenton et al. A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5'-3' mRNA exonuclease in yeast. Proc Natl Acad Sci USA. 2017 114:E9493-E9501. doi: 10.1073/pnas.1711680114.

TFIIH is a large nuclear complex of 10 subunits involved in DNA repair and transcription of protein-coding genes. Intriguingly, some of its subunits are not limited to their presence within TFIIH and can be detected in other complexes, in which they may have additional functions. This is well illustrated by the XPD subunits of TFIIH, which has been recently implicated in mitosis as well as in the mitochondria answer to oxidative stress. These observations prompt us to analyze whether other subunits of TFIIH are involved in these cellular processes. Understanding how the subunits of this complex are involved in various cellular mechanisms is of paramount importance, as mutations within TFIIH subunits give rise to rare human recessive disorders with complex and unexplained phenotypes.

Keywords: Transcription, DNA repair, Mitosis, rare diseases

Relevant publications:

Compe E, Pangou E, Le May N, Elly C, Braun C, Hwang JH, Coin F, Sumara I, Choi KW, Egly JM. Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions. Sci Adv. 2022 Aug 19;8(33):eabp9457. doi: 10.1126/sciadv.abp9457.

Sandoz J, Nagy Z, Catez P, Caliskan G, Geny S, Renaud JB, Concordet JP, Poterszman A, Tora L, Egly JM, Le May N, Coin F. Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression. Nat Commun. 2019 Mar 20;10(1):1288. doi: 10.1038/s41467-019-09270-2.

Compe E, Genes CM, Braun C, Coin F, Egly JM.  TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription. Nat Commun. 2019 May 7;10(1):2084. doi:10.1038/s41467-019-10131-1.

During development, distinct cell types acquire their properties under the control of transcription factors called cell fate determinants. These factors act on genes necessary for the progression of cell differentiation and this is accompanied by chromatin reorganisation. The acquisition of a specific chromatin conformation is required for cell differentiation and function. These two phenomena, gene regulation and chromatin reorganisation, have been well studied independently for numerous systems however their interdependency is poorly understood.

Our laboratory is studying the differentiation of glia in the developing Drosophila embryo to understand how the differentiation program controls chromatin reshaping. Our recent data identified four histone modifiers (Gcn5, Kdn4b, Gug and Su(var)3-9) directly induced by the glia fate determinant Gcm during the differentiation of the precursor into glia. The PhD candidate will characterise how these histones modifiers promote the differentiation of glial cells using the powerful genetic tools available in Drosophila combined with imaging (confocal microscopy) and state of the art sequencing technologies (ATACseq, Cut&Run, Cut&Tag).

The completion of this project will broaden our understanding of the chromatin ballet occurring during glial differentiation and will establish a clear link between the glial developmental program and the chromatin dynamics. In addition, given the conservation of most molecular pathways from Drosophila to mammals, this project will highlight the fundamental mechanisms controlling the development of the mammalian nervous system and will provide an experimental approach for their characterisation in other models.

Keywords: Drosophila, chromatin, gliogenesis, Cut and Run, high throughput sequencing

Relevant publications:

Sakr, R., P. B. Cattenoz, A. Pavlidaki, L. Ciapponi, M. Marzullo et al., 2022 Novel cell- and stage-specific transcriptional signatures defining Drosophila neurons, glia and hemocytes. bioRxiv: 2022.2006.2030.498263.

Pavlidaki, A., R. Panic, S. Monticelli, C. Riet, Y. Yuasa et al., 2022 An anti-inflammatory transcriptional cascade conserved from flies to humans. Cell Rep 41: 111506.

Cattenoz, P. B., A. Popkova, T. D. Southall, G. Aiello, A. H. Brand et al., 2016 Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila. Genetics 202: 191-219.

Title: identifying distinct ATRA-dependent populations of spermatogonia in the mouse testis

Description: We are deciphering the physiological role played by retinoic acid (ATRA) nuclear receptors (RAR), by using a large variety of techniques including genetics, molecular biology, imaging and biochemistry (1). We use the seminiferous epithelium (SE) of the mouse testis as a model system because ATRA is indispensable to spermatogonia (SG) differentiation (2). The aim of the project is to test the hypothesis that distinct populations of ATRA-dependent SG coexist in the SE, and to identify the gene networks that are controlled by ATRA. This will explain why and how spermatogenesis perpetuates for months in the SE of mice lacking either RARG or RARA, but is arrested when both of them are lacking (3). To do so, whole post-natal day 5 (PND5) and PND8 testis cell suspensions were made from mice lacking all ATRA-synthesizing enzymes in their SE (4). Then, both chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) were determined on 10 000 nuclei of each by using the “single cell multiome” approach developed by 10X Genomics. The candidate will use these datasets, as well as publicly available datasets on RAR-binding sites genome-wide (5), to identify the genes controlled by ATRA-activated RARs at PND5 and PND8 in the SE. This corresponds to the time period during which at least 2 distinct populations of SG are sensitive to different ATRA-dependent signals.

(1) Mark et al., (2006) Annu. Rev. Pharmacol. Toxicol. 46:451;

(2) Teletin et al., (2017) Curr. Top. Dev. Biol. 125:191;

(3) Gely-Pernot et al., (2015) PLoS Genet. 11:e1005501;

(4) Teletin et al., (2019) Development 146:dev170225

(5) Chatagnon et al., (2015) Nucleic Acids Res. 43:4833

Keywords: nuclear receptors, gene regulation (RNA-seq, single cell), epigenetics (ATAC-seq), cell differentiation, cell morphogenesis, stem cell niche, mouse genetics, bioinformatics

The maintenance, integrity and proper expression of the plant mitochondrial genome (mtDNA) are fundamental for plant fitness and survival, and are under the control of nuclear-encoded factors that are targeted to mitochondria. We have recently characterized the plant-specific 5'-3' exonuclease OEX1, whose loss has dramatic effects on mtDNA genome stability and plant development. OEX1 has preference for RNA:DNA hybrids and is likely responsible for the degradation of Okazaky primers during mtDNA replication, as well as for the elimination of R-loops that compromise genome stability. It further has Flap-endonuclease activity, implying additional roles in base excision repair and the processing of recombination intermediates. In animal mitochondria, the same functions are accomplished by mito-targeted RNase H and Flap-endonuclease FEN1. In addition to OEX1, an RNase H and FEN1 have also been found in plant mitochondria, and the interplay of these apparently redundant proteins in plant mtDNA maintenance is not understood.

We propose to study the specific functions of these nucleases in plant mtDNA replication, recombination and repair. This will be done using available Arabidopsis thaliana mutants, genetic complementation and co-immunoprecipitation approaches, coupled to DNA-seq analysis of mtDNA replication and stability. The replication of a specific mitochondrial episome that is a good model of mtDNA replication will be followed, using biochemical approaches. We will also study the interplay with the corresponding OEX and RNase H chloroplast homologs.

Keywords:

Mitochondrial genome, Arabidopsis, recombination, exonucleases, RNase

1. Chevigny N., Weber-Lotfi F., Le Blevenec A., Nadiras C.; Fertet A., Bichara M., Erhardt M., Dietrich A., Raynaud C. and Gualberto J.M. (2022). RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest. PLoS Genet., e1010202. doi: 10.1371/journal.pgen.1010202.
2. Fertet A., Graindorge S., Koechler S., De Boer G.J., Guilloteau-Fonteny E. and Gualberto J.M. (2021). Sequence of the mitochondrial genome of Lactuca virosa suggests an unexpected role in Lactuca sativa’s evolution. Frontiers in Plant Science, 12. doi: 10.3389/fpls.2021.697136
3. Gualberto J.M. and Newton K.J. (2017). Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu. Rev. Plant Biol., 68: 225-252

Our group has characterized a novel innate immunity pathway functioning in the control of viral infections in the model organism drosophila. This pathway involves the evolutionarily conserved molecule STING, which activates an NF-kB-dependent transcriptional program that mediates resistance to viral infection. We have recently identified two cyclic GMP-AMP synthase (cGAS)-like receptors (cGLR1 and 2) involved in the activation of this pathway, upon detection of viral infection. Unexpectedly, experiments using recombinant proteins or extracts from transfected mammalian HEK293 cells suggest that cGLR1 and cGLR2 make different cyclic dinucleotides (CDNs). While they both produce 3′2′-cGAMP, cGLR1 also produces 2′3′-c-di-AMP whereas cGLR2 produces 2′3′-cGAMP. Although 3′2′-cGAMP is the best agonist for drosophila STING, 2′3′-cGAMP activates the pathway and our preliminary results indicate that 2′3′-c-di-AMP is also active in flies. The PhD project aims at combining drosophila genetics and state of the art mass spectrometry techniques to detect the CDNs produced in vivo in whole fly extracts or circulating in hemolymph from wild-type or cGLR mutant flies in response to viral infection. In a second step, the function of these CDNs will be studied in flies, with a special focus on their potential function as immunotransmitters.

Keywords: innate immunity/drosophila/virus/STING/cGAS/genetics

• Schneider J & Imler JL (2021) Sensing and signalling viral infection in drosophila. Developmental and Comparative Immunology, 117: 103985.
• Cai H & Imler JL (2021) cGAS-STING: insight on the evolution of a primordial antiviral signaling cassette. Faculty Reviews, 10: (54).
• Cai H, Meignin C & Imler JL (2022) cGAS-like receptor-mediated immunity: the insect perspective. Current Opinion in Immunology,74: 183-189.

The ribosome is the macromolecular complex in the cell that translates the genetic code specified in messenger RNA into proteins. Our team has been studying this process for many years by determining structures of functional complexes of the ribosome with various ligands and protein factors using both X-ray crystallography and single particle cryo-electron microscopy techniques. Because protein biosynthesis is pivotal for all life, it is an extremely controlled process being regulated in many ways by numerous pathways, ligands, and factors. One way of gaining control of translation is by modifying the rRNA parts of the ribosome and thereby altering their function.

Emerging studies indicate that variations in 2’-O-methylation of rRNA are vital for control of protein expression during development, and they have also been implicated in cancer where modified ribosomes can be tuned for infinite cellular growth. By determining cryo EM ribosome structures taken at different well-defined developmental stages from Caernorhabditis elegans, we will characterize the patterns of 2’-O-methylations which will allow us to decipher the role of these modifications from the apparent effect on ribosome structure in order to understand their involvement in regulation of translation during cell development and differentiation.

Keywords: Structural Biology, Ribosomes, Translation, cryo EM, RNA modifications

Relevant publications:

• Djumagulov M, Demeshkina N, Jenner L, Rozov A, Yusupov M, Yusupova G. Accuracy mechanism of eukaryotic ribosome translocation. Nature. 2021 Dec;600(7889):543-546. doi: 10.1038/s41586-021-04131-9. Epub 2021 Dec 1. PMID: 34853469; PMCID: PMC8674143.
• Zgadzay Y, Kolosova O, Stetsenko A, Wu C, Bruchlen D, Usachev K, Validov S, Jenner L, Rogachev A, Yusupova G, Sachs MS, Guskov A, Yusupov M. E-site drug specificity of the human pathogen Candida albicans ribosome. Sci Adv. 2022 May 27;8(21):eabn1062. doi: 10.1126/sciadv.abn1062. Epub 2022 May 25. PMID: 35613268; PMCID: PMC9132455.
• Flygaard RK, Boegholm N, Yusupov M, Jenner LB. Cryo-EM structure of the hibernating Thermus thermophilus 100S ribosome reveals a protein-mediated dimerization mechanism. Nat Commun. 2018 Oct 9;9(1):4179. doi: 10.1038/s41467-018-06724-x. PMID: 30301898; PMCID: PMC6177447.

Structural investigation of RNA polymerase and its cooperation with other molecular machines

Gene expression employs at least two steps in all kingdoms of life: i) mRNA is transcribed from DNA by RNA polymerase (RNAP); and ii) mRNA is translated to protein by the ribosome; The machineries executing gene expression have been mostly studied in isolation in the past but we know many of them are organized in large, supramolecular assemblies where their activities are coordinated and where new functions emerge not predictable from the individual components. We aim to close this gap. For example, we study the expressome, a supramolecular assembly line where RNAP is coupled to the ribosome to understand how the two key players in gene expression cooperate with each other (see Webster et al., Science 2020 for recent result). We also would like to understand how transcription termination occurs through the interplay of RNAP with the termination factor Rho in pathogenic bacteria with the potential to identify new targets for anti-bacterial drugs.

Students interested to tackle challenging problems and using state-of-the-art structural biology approaches, will be able to choose from a number of projects and study the allosteric crosstalk between RNAP and other key enzymes involved in bacterial gene expression.

Interested candidates will use single particle cryo-EM, the ideal method to gain mechanistic insights of large, dynamic protein nucleic acid complexes, to obtain high-resolution reconstructions. Biochemical, and in-vivo approaches will complement your results. Your work will unravel how synergism and crosstalk of distinct macromolecular machines in the context of a supramolecular assembly regulates the conversion of genotype to phenotype.

Key words: RNA polymerase, ribosome, molecular machines, gene expression, cryo-electron microscopy, structural biology, transcription termination

References:

Dey S, Batisse C, Shukla J, Webster MW, Takacs M, Saint-André C, and Weixlbaumer A (2022). Structural insights into RNA-mediated transcription regulation in bacteria.  Mol Cell 82(20), 3885–3900.

Webster MW, Takacs M, Zhu C, Vidmar V, Eduljee AD, Abdelkareem M,  Weixlbaumer A (2020). Structural basis of transcription-translation coupling and collision in bacteria. Science 369(6509), 1355-1359

Abdelkareem M, Saint-André C, Takacs M, Papai G, Crucifix C, Guo X, Ortiz J, Weixlbaumer A (2019). Structural Basis of Transcription: RNA polymerase backtracking and its reactivation. Mol Cell 75(2), 298-309

Deciphering the (ultra-) structural mechanisms of Golgi stacking

Proper glycosylation of proteins in the endomembrane system is crucial for many biological processes, such as lysosomal sorting, extracellular matrix structuring, and signal transduction. Improper glycosylation, in turn, is associated with neurodegeneration, cancer, and autoimmune diseases. It is, thus, vital for cells to maintain the order of their central glycan modification hub, the Golgi apparatus. In this context, the Golgi matrix, a dense protein assembly, guides protein distribution amongst the individual Golgi cisternae, controls their shape and keeps them stacked. Nevertheless, how the Golgi matrix is structured to achieve these functionalities remains largely unknown, as the usability of classical approaches based on fluorescence microscopy, room temperature electron microscopy, and in vitro reconstitution is limited by the small size and high complexity of the system. To overcome this, we will employ state-of-the-art in situ cryo-electron tomography combined with subtomogram averaging to visualize the Golgi matrix and its proteins at (sub-) nanometer resolution in its intracellular environment. This will provide an (ultra-) structural framework for the integration of orthogonal data provided by reverse genetics, cell biology, and biochemistry techniques. Together, we will use these approaches to provide unprecedented insights into the structure-function relationship of the Golgi apparatus organization.

Working on this project, you will learn:

• to employ CRISPR-based technologies for genetic engineering and light microscopy techniques for the phenotypic characterization of cell lines.
• to perform complete in situ structural biology workflows, including cryo-electron tomography, from specimen preparation via data acquisition and image processing to model fitting.
• to use and build software tools for the quantitative computational analysis of three-dimensional ultrastructural data to elucidate principles of cellular organization
• to develop the scientific independence to conduct and present an elaborate, cross-disciplinary project.

Relevant publications:

Florian Fäßler, Manjunath G. Javoor, Julia Datler, Hermann Döring, Florian W. Hofer, Georgi Dimchev, Victor-Valentin Hodirnau, Klemens Rottner & Florian K.M. Schur. ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning.bioRxiv 2022.07.28.501813, https://doi.org/10.1101/2022.07.28.501813

Florian Fäßler, Georgi Dimchev, Victor-Valentin Hodirnau, William Wan & Florian K.M. Schur. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.Nature Communications volume 11, Article number: 6437, 2020, https://doi.org/10.1038/s41467-020-20286-x

Florian Fäßler, Bettina Zens, Robert Hauschild & Florian K.M. Schur. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology, Volume 212, Issue 3, 2020.

The 3D organisation of the genome is essential for cellular homeostasis and organism development. The Cohesin complex is a major actor of this 3D organisation by participating in sister chromatid cohesion, regulation of transcription and formation of chromatin loops. Mutations affecting Cohesin lead to numerous cancers and neurodevelopmental disorders.

Cohesin is formed of a tripartite core complex (SMC1A/SMC3/RAD21) and its functions throughout the cell cycle are driven by its ATPase activity and the interaction of the complex with several regulators. Our team recently revealed the molecular basis of the Cohesin ATPase cycle and how it underlies the Cohesin mode of action. We also started to unveil the in vivo consequences of perturbed cohesin regulation on early zebrafish development.

To follow up on our recent discoveries, the PhD candidate will perform in vitro and in vivo studies to characterize the effect of specific functional and disease mutants of SMC1A, SMC3 and RAD21 on the mode of action (ATP and DNA binding, ATP hydrolysis, interactions with regulators) of Cohesin and their consequences on embryonic development in zebrafish. The 3-D architecture of the genome will be also studied to determine the impact of these mutants on the overall chromatin organization.

Keywords: genome organization, transcription, chromatin, Cohesin, ATPase activity, biochemistry, human disease modelling, zebrafish.

• Vitoria Gomes, M., et al., Specific conformational dynamics of the ATPase head domains and DNA exit gate mediate the Cohesin ATPase cycle. bioRxiv, 2022: p. 2022.06.24.497451.
• Ramos-Morales, E., et al., The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination. Nucleic Acids Res, 2021. 49(11): p. 6529-6548.
• Marek, M., et al., Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2. Cell Rep, 2021. 37(12): p. 110129.

Myc is a master transcription activator that directly stimulates the expression of 15% of the human genome and is one of the most frequently deregulated driver gene in human cancers. Among other tumors, Myc activity is deregulated in Rhabdoid cancer, a very aggressive childhood cancer with low survival rates. In patients with Rhabdoid cancer the protein Snf5 is mutated or absent as large parts of its gene, or even the whole gene, are deleted. Snf5 is a subunit of the chromatin remodeling complex Swi/Snf. It was shown that Snf5 interacts with the DNA binding pocket of Myc and attenuates in this manner the transcriptional activity of Myc. When Snf5 is deleted, as in Rhabdoid cancer, Myc is no longer held in check, yielding a destructive alteration to the transcription program of the cell.

To understand the molecular background of this process, the goal of this project is to reconstitute the assembly between Swi/Snf and Myc, and to solve its structure using cryo-Electron Microscopy. Our team has already developed human cell lines with affinity-tagged subunits of Swi/Snf that will be instrumental in purifying this complex. Potentially, the structure will serve to guide development of anti-cancer drugs that mimic the way Snf5 blocks Myc binding to DNA.

Interested candidates will be trained for purification of low-abundance complexes from human cells, functional assays and atomic structure determination by single particle cryo-EM.

Keywords: Chromatin remodelers, Myc oncogene, Structural biology, cryo-electron microscopy, cancer

Relevant publications:

• Papai, G., Frechard, A., Kolesnikova, O., Crucifix, C., Schultz, P., and Ben-Shem, A. (2020) Structure of SAGA and mechanism of TBP deposition on gene promoters, Nature 577, 711-716.
• Kolesnikova O, Ben Shem A, Luo J, Ranish J, Schultz P, Papai G (2018) Molecular structure of promoter-bound yeast TFIID Nature communications 9: 4666
• Sharov G, Voltz K, Durand A, Kolesnikova O, Papai G, Myasnikov AG, Dejaegere A, Ben Shem A, Schultz P (2017) Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA. Nature communications 8: 1556

Tomographic reconstructions obtained from flash frozen samples represent the ultimate quality of the structural information of cell organization available to date, reaching near-atomic resolution. However, the information extraction from cryo-electron tomography (cryo-ET) reconstructions remains challenging particularly because of a high noise and deformations due to a limited tilt angular range accessible in cryo-ET. Emerging approaches based on computer vision and deep learning offer new opportunities to overcome these challenges to take full advantage of cryo-ET for understanding biological functions.

Recent work by the team has demonstrated the value of combining cryo-ET and deep learning denoising approaches to annotate nucleosomes and DNA linkers in their functional environment of the cell nucleus, preserved in a near-native state. The aim of this project is to further develop this methodological synergy by developing new computational tools based on deep learning and involving 3D segmentation, pose estimation and deformable image registration to extract information from chromatin tomograms at three structural levels: organization of chromatin domains, local geometry of nucleosome fibers and nucleosome conformation. Comparative analysis of the local structural landscapes of active and inactive chromatin will reveal mechanisms of epigenetic regulation based on chromatin structure.

Keywords: cryo-electron tomography, data mining, artificial intelligence, deep learning, computer vision, chromatin, epigenetics

Relevant publications:

1. Fatmaoui F., Carrivain P., Grewe D., Jakob B., Victor JM., LeforestierA., Eltsov M. Cryo-electron tomography and deep learning denoising reveal native chromatin landscapes of interphase nuclei. bioRxiv 2022.08.16.502515; doi: 10.1101/2022.08.16.502515

2.  Harastani M., Eltsov M., Leforestier A., Jonic S. TomoFlow: Analysis of Continuous Conformational Variability of Macromolecules in Cryogenic Subtomograms based on 3D Dense Optical Flow. J Mol Biol. 2022;434(2):167381. doi: 10.1016/j.jmb.2021.167381

3. Harastani M., Eltsov M., Leforestier A., Jonic S. HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes. Front Mol Biosci. 2021 8:663121. doi: 10.3389/fmolb.2021.663121.

Nuclear receptors (NR) are the largest family of transcription factors that regulate the transcription of genes in metazoans. They control many processes related to cell cycle, differentiation, apoptosis, development, reproduction and homeostasis. An important feature of NRs is that their regulation of gene expression is dependent on the fixation of small molecule ligands. This ligand dependency makes them important targets for drug development in many diseases such as diabetes, arteriosclerosis, inflammatory diseases, cancer, etc.

Genetic alterations to members of the nuclear receptor family are associated to various cancers, which underscores the necessity to better understand the molecular mechanism of action and the effects of specific mutations linked to carcinogenesis.

The aim of the PhD project is to investigate the structure-dynamic-function relationships of PPARg and RXRa mutations and molecular mechanisms of interaction and to characterize the changes in structural dynamics induced by cancer associated mutations. Innovative simulation approaches and state of the art analysis methods will be employed. We expect that this project will have an impact by improving our understanding of the effect of carcinogenic mutations of PPAR, which could contribute to improving anti-cancer treatments by facilitating the development of molecules that might be used to modulate PPAR action in pathological situations.

Keywords: nuclear receptors, cancer, molecular dynamics simulations, molecular modeling

Relevant publications:

• Rochel N, Krucker C, Coutos-Thévenot L, Osz J, Zhang R, Guyon E, Zita W, Vanthong S, Hernandez OA, Bourguet M, Badawy KA, Dufour F, Peluso-Iltis C, Heckler-Beji S, Dejaegere A, Kamoun A, de Reyniès A, Neuzillet Y, Rebouissou S, Béraud C, Lang H, Massfelder T, Allory Y, Cianférani S, Stote RH, Radvanyi F, Bernard-Pierrot I. Recurrent activating mutations of PPARγ associated with luminal bladder tumors. Nat Commun. 2019 Jan 16;10(1):253. doi: 10.1038/s41467-018-08157-y. PMID: 30651555; PMCID: PMC6335423.
• Eberhardt J, Stote RH, Dejaegere A. Unrolr: Structural analysis of protein conformations using stochastic proximity embedding. J Comput Chem. 2018 Nov 15;39(30):2551-2557. doi: 10.1002/jcc.25599. PMID: 30447084.
• Chebaro Y, Sirigu S, Amal I, Lutzing R, Stote RH, Rochette-Egly C, Rochel N, Dejaegere A. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ. PLoS One. 2017 Jan 26;12(1):e0171043. doi: 10.1371/journal.pone.0171043. PMID: 28125680; PMCID: PMC5268703.

In eukaryotes, DNA is made to fit inside the cell nucleus through a high degree of
compaction that is enabled by assembly into chromatin. Processes such as DNA damage
repair and transcription require localized changes in chromatin compaction. Reorganization
of nucleosomes to regulate accessibility is mediated by a set of multi-subunit
ATP-dependent chromatin remodeling complexes that slide or evict nucleosomes from
the chromatin fiber. Deregulation of these complexes can severely impact gene
expression, cell identity and genome integrity. The mammalian SWI/SNF complex
comprises a set of evolutionary conserved ‘core and enzymatic’ subunits, but also
‘auxiliary’ subunits present only in mammalians, thought to reflect increasing biological
complexity. Although the topology and 3D structure of the SWI/SNF complex are
becoming clearer, the structure, the molecular details of interaction and the function of
these auxiliary yet important subunits are poorly defined at best. Despite some evidence
that certain auxiliary subunits are involved in DNA damage repair (DDR), our knowledge
of their actual function in this process is very limited. Intriguingly, several of these
proteins are predicted to be intrinsically disordered (ID), suggesting they may undergo
liquid-liquid phase separation (LLPS). By using a combination of cryo-EM, ,
biochemistry and live imaging experiments, this project aims to define the structure and
function of auxiliary subunits of the mammalian SWI/SNF complex, determine how they
interact with the core subunits and with the nucleosome and better understand their roles
at sites of DNA damage, keeping into consideration their potential for phase separation.

Keywords: Chromatin remodeling, Epigenetic, Cryo-EM, live cell imaging, DNA-damage

Relevant publications:
1. Molecular basis for the methylation specificity of ATXR5 for histone H3. Elisa Bergamin, Sarvan S, Mallette J, Eram MS, Yeung
S, Mongeon V, Joshi M, Brunzelle JS, Michaels SD, Blais A, Vedadi M, Couture JF. Nucleic Acids Research. 2017
2. Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Jacob Y*, Elisa Bergamin*, Donoghue
MT, Mongeon V, LeBlanc C, Voigt P, Underwood CJ, Brunzelle JS, Michaels SD, Reinberg D, Couture JF, Martienssen RA. Science. 2014. * denotes co-first authorship.
3. The Cytoplasmic Adapter-Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization. Elisa Bergamin, Peter T. Hallock, Steven J. Burden, and
Stevan R. Hubbard. Molecular Cell. 2010, 39: 100-109.

Structural investigations on Rea1 complexes

Eukaryotic ribosomes are complex molecular machines whose assembly is tightly controlled to ensure faithful protein biosynthesis. Ribosome assembly is initiated in the nucleolus by the transcription and processing of ribosomal RNAs, which together with ribosomal proteins form pre-60S ribosomal particles. The pre-60S particles mature by transiently interacting with various assembly factors during cytosolic export. The nearly 5000 residues long maturation factor Rea1 is vital for the export of the pre-60S particles. Rea1 belongs to the AAA+ protein family and harnesses the energy of ATP hydrolysis to mechanically remove assembly factors from pre-60S particles. Despite its key importance for ribosome maturation, the Rea1 structure and mechanism are poorly understood. We will carry out genetic manipulations in yeast to tag proteins for subsequent Rea1-pre-60S purification. The PhD student will also be trained in state-of-the-art cryo-electron microscopy to structurally characterize these complexes. There are already preliminary structural data on a Rea1-pre-60S complex providing a solid starting ground for the PhD project.  These structural investigations will be complemented by in-vitro as well in-vivo activity assays carried out in collaboration with external partners. He/she will also learn to work in a highly international environment and to develop project management skills. Furthermore, presentation skills will be acquired due to regular progress meetings and the attendance of international conferences.

Key words: Rea1, molecular machines, ribosome maturation, AAA+ proteins, cryo-electron microscopy, structural biology

Relevant publications:

Sosnowski P, Urnavicius L, Boland A, Fagiewicz R, Busselez J, Papai G, Schmidt H. The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1. Elife 2018.

Schmidt H, Zalyte R, Urnavicius L, Carter AP. Structure of human cytoplasmic dynein-2 primed for its power stroke. Nature 2015.

Schmidt H, Gleave ES, Carter AP. Insights into dynein motor domain function from a 3.3-A crystal structure. NSMB 2012.

Title: Molecular Investigations of Human Splicing Fidelity Checkpoints

The spliceosome removes non-coding introns from precursor messenger RNAs during pre-mRNA splicing in eukaryotes. Crucially, many pre-mRNAs are spliced differently depending on cellular status or external stimuli. This “alternative splicing” reshapes the genetic information from a given mRNA to encode several protein isoforms and greatly diversifies proteomes.

Splicing must be extremely precise as errors produce aberrant mRNA encoding potentially toxic proteins. Splicing fidelity relies on the spliceosome's accuracy, which defines the introns' boundaries before catalysing their excisions.

This project consists of a mechanistic study of the human spliceosome aimed at unravelling how it ensures the fidelity of pre-mRNA splicing. The PhD student will use a combination of biochemical and structural biology approaches to reveal the molecular organisation of splicing complexes captured during key fidelity checkpoints. These complexes contain numerous protein and RNA subunits and typically require a wide array of methods to be generated, and then, precisely characterised. In particular, the PhD student will have the opportunity to use ribonucleoprotein complexes reconstitution from cell extracts and/or recombinant sources, various biochemical and biophysical assays, cryo-EM, X-ray crystallography, mass spectrometry…

Keywords: Spliceosome Ribonucleoprotein Structure Splicing Fidelity cryo-EM

Relevant publications:

Charenton, C.*†, Wilkinson, M.E.*†, Nagai, K.*, 2019. Mechanism of 5′ splice site transfer for human spliceosome activation. Science 364, 362–367.

Wilkinson, M.E.*†, Charenton, C.*†, Nagai, K.†, 2020. RNA Splicing by the Spliceosome. Annual Review in Biochemistry 89, 359–388.

Plaschka, C., Lin, P.-C., Charenton, C., Nagai, K., 2018. Prespliceosome structure provides insights into spliceosome assembly and regulation. Nature 559, 419–422.

RNA virus infection in plants depends on the ability of the virus to replicate its RNA genome and to transport the replicated viral RNA from the infected cell into adjacent cells through pores in the plant cell walls called plasmodesmata (PD). Plant RNA viruses and dsRNA produced during virus replication or applied externally induce pathogen-triggered immunity (PTI) responses (MAPK activation, ethylene induction), including the formation of callose deposits at PD, thus leading to PD closure and inhibiting virus movement. This indicates that viral nucleic acids are pathogen/virus associated molecular patterns (PAMP/VAMP) that trigger PTI in addition to RNA silencing. Importantly, emerging studies indicate that virus-encoded movement proteins (MPs) act as pathogen effectors that suppress the dsRNA-induced immunity response at PD to facilitate viral RNA spread between cells. In the PhD thesis, the PhD student will use molecular approaches in proteomics, genetics, cell biology, and virology to identify the mechanism through which MP effectors suppress dsRNA-induced immunity at the PD and facilitate virus movement and infection. 

Keywords: plant immunity, dsRNA, pattern-triggered immunity, plant virus, plasmodesmata, movement protein, proteomics, plant virology, plant cell biology, plant genetics

• Huang C, Sede AR, Elvira-Gonzalez L, Yan Y, Rodriguez M, Mutterer J, Boutant E, Shan L, Heinlein M (2022) dsRNA-induced immunity targets plasmodesmata and is suppressed by viral movement proteins. bioRxiv; https://doi.org/10.1101/2022.11.21.517408 (revised version under review, Plant Cell)
• Niehl A, Wyrsch I, Boller T, Heinlein M (2016) Double-stranded RNA induces a pattern-triggered immune signalling pathway in plants. New Phytol 211, 1008-1019
• Kørner CJ, Pitzalis N, Peña EJ, Erhardt M, Vazquez F, Heinlein M (2018) Crosstalk between PTGS and TGS pathways in natural antiviral immunity and disease recovery. Nat Plants 4, 157-164

Pervasive transcription, whereby transcripts get spuriously generated from all over the genome, often in-antisense to functional genes, is a universal and ambivalent property of living systems. Antisense transcription can be undesirable and even dangerous as it contributes to gene expression noise and may lead to genome instability. But it also conceals an inexhaustible source of evolutionary innovation, from which new functional RNAs and regulatory circuits can arise. Two conserved RNA-binding proteins (RBPs), transcription termination factor Rho and double-stranded RNA-specific RNase III, function at co- and post-transcriptional levels to restrain pervasive transcription and even "adopt" some of the resulting RNAs for regulatory purposes in bacteria. Therefore, these RBPs are potentially important evolutionary factors that, by "managing" the transcriptome, hold keys to bacterial adaptation and long-term success. To understand the evolutionary significance of pervasive antisense transcription and of the RBPs that control it, we will perform large-scale laboratory evolution experiments involving two phylogenetically and ecologically distant bacteria, a human commensal Escherichia coli and the pathogen Staphylococcus aureus. We will see how both bacteria, deprived of Rho or RNase III, adapt, under various conditions, to unleashed pervasive transcription and other unique gene expression challenges caused by this handicap. Leveraging the complementary expertise of both laboratories in RNA biology of Gram-positive and Gram-negative bacteria, using genomic, transcriptomic, genetic, biochemical, and physiological approaches, we will address, in a comparative and synthetic way, the underlying molecular mechanisms and the adaptive rationale of the observed evolutionary events.

Key words: pervasive transcription, antisense RNA, RNA-binding protein, Rho, RNase III, bacteria, Escherichia coli, Staphylococcus aureus, experimental evolution

Muscle genetic diseases affect children and adults in all populations, and represent a significant burden for the patients, their families and public health care. They can be associated with muscle weakness, muscle pain, breathing difficulties, and delayed motor milestones, and are potentially lethal. Several causative genes are known. However, almost half of patients do not have a molecular diagnosis, precluding genetic counseling and specific healthcare, and indicating the implication of yet unknown genes. We previously performed exome, genome and RNA sequencing on more than 500 affected individuals and their families, and this has led to the identification of several causative genes. The PhD project aims to combine bioinformatics analysis of our sequence database with functional assays in vitro and in vivo to identify and validate novel genes mutated in muscle diseases. Through an in-house developed bioinformatics pipeline, the PhD student will handle and filter the large datasets to identify best candidate genes. Segregation analysis and the impact of mutations on the RNA integrity will be assessed in vitro. The most convincing genes will subsequently undergo functional tests in cell models to investigate the effect of mutations on the function of the protein and gain a first insight into the pathomechanism. Then, to prove that the mutation is at the origin of the muscle disease, the PhD student will establish transient (viral transduction) or stable (knock-in or knock-out transgenesis) mouse models to characterize disease development. The identification of the genetic causes of muscle diseases will significantly improve molecular diagnosis and genetic counseling, contribute to a better understanding of skeletal muscle function under normal and pathologic conditions, and uncover potential targets for prospective therapeutic approaches.

Keywords: Congenital myopathies, myalgia, human genetics, genome sequencing, RNA sequencing, cellular models, mouse models

Relevant publications:

- Lornage X*, Romero NB*, Grosgogeat CA#, Malfatti E#, Donkervoort S, Marchetti MM, Neuhaus SB, Foley AR, Labasse C, Schneider R, Carlier RY, Chao KR, Medne L, Deleuze JF, Orlikowski D, Bönnemann CG, Gupta VA, Fardeau M, Böhm J^, Laporte J^. ACTN2 mutations cause "Multiple structured Core Disease" (MsCD). Acta Neuropathol. 2019 Mar;137(3):501-519. doi: 10.1007/s00401-019-01963-8 (X Lornage = previous PhD student)

- Lionello VM, Nicot AS, Sartori M, Kretz C, Kessler P, Buono S, Djerroud S, Messaddeq N, Koebel P, Prokic I, Herault Y, Romero NB, Laporte J*, Cowling BS*. Amphiphysin 2 (BIN1) modulation rescues MTM1 centronuclear myopathy and prevents focal adhesion defects. Sci Transl Med. 2019 Mar 20;11(484). doi: 10.1126/scitranslmed.aav1866. (V Lionello = previous PhD student) 

- Gómez-Oca R, Edelweiss E, Djeddi S, Gerbier M, Massana-Muñoz X, Oulad-Abdelghani M, Crucifix C, Spiegelhalter C, Messaddeq N, Poussin-Courmontagne P, Koebel P, Cowling BS^, Laporte J^. Differential impact of ubiquitous and muscle dynamin 2 isoforms in muscle physiology and centronuclear myopathy. Nat Commun. 2022 Nov 11;13(1):6849. doi: 10.1038/s41467-022-34490-4. (R Gomez-Oca = previous PhD student) 

Deciphering the role of Rbmx and its retrocopy Rbmxl1 in the developing brain in humans and mice

Proper development and functioning of the cerebral cortex depend on the coordinated production, migration and differentiation of neurons. Accordingly, disruption of one or several of these cellular processes can cause neurodevelopmental disorders (NDD). The identification of the molecular and cellular mechanisms that control cortical development is crucial to better understand NDD. RBMX is a gene located on chromosome (chr) X in mammals that encodes hnRNP G (Heterogeneous nuclear ribonucleoprotein G), a multifunctional protein able to regulate expression and splicing of other genes as a component of the spliceosome. Notably, pathogenic variants in RBMX are associated with a severe neurodevelopmental disorder characterized by intellectual disability, brain and eye malformations affecting hemizygous males (preliminary results). RBMX has several retrocopies located on autosomes. The most recent retrocopy, RBMXL1 encode likely functional proteins, 96-98% identical to RBMX/Rbmx, expressed during brain development in humans and mice. The reason why Rbmx has been several times retrotransposed during evolution and how these retrocopies have shaped brain evolution in mammals is fully unknown so far. Genetic findings in humans, mouse, zebrafish and xenopus all support a highly conserved role of RBMX in controlling brain development. Importantly, all these pioneer studies on RBMX function are not differentiating RBMX and RBMXL1, and therefore are likely reflecting the function of both RBMX and RBMXL1 without investigating potential convergent or divergent functions. Further investigations are therefore needed to identify function of RBMXL1 and its redundancy with RBMX.

In this ANR-funded project, our specific objectives are first to dissect the neuronal function(s) of Rbmx and Rbmxl1 and second to confirm and disentangle the probable functional redundancy existing between Rbmx and Rbmxl1 retrocopy using both mouse models and human iPSCs. As our preliminary data strongly indicate a functional redundancy between Rbmx and Rbmxl1, it is critical to address these questions in both mouse models invalidated for Rbmx or Rbmxl1 individually or concomitantly. We will dissect both the cellular (histology and live imaging of progenitor biology) and the molecular (RNAseq) mechanisms by which RBMX and its functional retrocopies contribute to brain development.

Keywords: human genetic disorder, brain development, RBMX, retrocopy, functional redundancy

Relevant publications:

1/ Asselin L, et al. Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity. .Nature Communication (2020) 11: 2441

2/ Ramos-Morales E et al. The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination. Nucleic Acids Res (2021) 49: 6529-6548

3/ Laguesse S et al. A Dynamic Unfolded Protein Response Contributes to the Control of Cortical Neurogenesis. Developmental Cell (2015) 35: 553-67

Control of protein synthesis is a fine-tuned process that involves a large panel of RNA binding proteins (RBPs). Mutation or loss of expression of RBPs is a main cause of neurodevelopmental diseases. Fragile X syndrome (FXS) is the first cause of familial intellectual disability and is due to the loss of the RBP FMRP. FMRP interacts with mRNA targets to repress or activate their translation by unknown mechanism(s). We previously found that DGKk mRNA is a preferred target of FMRP in mouse neurons (1) and DGKk expression is strongly decreased in the brain of FXS patients (2). DGKk (diacylglycerol kinase kappa) is a master regulator of neuronal signalling pathways that are deregulated in several types of intellectual disabilities. We provided evidence that DGKk loss of function plays an important role in the pathomechanism of the disease : DGKk loss of expression (Dgkk-KO mouse) reproduces phenotypes of the FXS mouse model (Fmr1-KO) and reexpression of DGKk in the brain of Fmr1-KO mouse is sufficient to correct its main deficits (2). Better understand how DGKk expression is controlled by FMRP will help elucidate how FMRP controls neuronal protein synthesis and how DGKk function contributes to neuronal signaling in pathological and normal condition.

Based on cross-linking immunoprecipitation methods (CLIP) coupled to RNA-seq performed in neurons, the FMRP binding site on DGKk will be delineated. Using a combination of techniques including gene reporter assays, site directed mutagenesis, polysome profiling, we will define the molecular rules of FMRP-dependent control. The PhD student will also contribute to the study of DGKk regulome and the development of a DGKk gene-based strategy for FXS therapy.

1. Tabet,R., Moutin,E., Becker,J.A., Heintz,D., Fouillen,L., Flatter,E., Krezel,W., Alunni,V., Koebel,P., Dembele,D., et al. (2016) Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons. Proc Natl Acad Sci U S A, 113, E3619-28.

2. Habbas,K., Cakil,O., Zámbó,B., Tabet,R., Riet,F., Dembele,D., Mandel,J.-L., Hocquemiller,M., Laufer,R., Piguet,F., et al. (2022) AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model. EMBO Mol Med, 14, e14649.

Keywords:

Translation control, FMRP, RNA-protein interaction, neurodevelopmental disorder

We are seeking a motivated PHD student to investigate how peculiar genetic mutations, namely microsatellite expansions, located in genomic regions annotated as “non coding” (5’ and 3’UTRs, introns, LncRNA, etc.) cause devastating muscle and/or neurodegenerative pathologies. We are notably interested in the following diseases: Fragile X Tremor Ataxia Syndrome (FXTAS), Neuronal Intranuclear Inclusion Disease (NIID), OcculoPharyngoDistal Myopathy (OPDM) and Amyotrophic Lateral Sclerosis (ALS).

FXTAS, NIID and OPDM are rare muscle and neurodegenerative diseases caused by GGC repeats located in different genes. Our work shows that despite being localized in “non-coding” sequences, these repeats are nonetheless translated into novel and toxic proteins (Sellier et al., Neuron 2017, Boivin et al., Neuron 2021, etc.).

ALS is the 3rd most common neurodegenerative disease worldwide. ALS is characterized by degeneration of motor neurons leading to muscle wasting and weakness, ultimately resulting in death of the patients. The most common genetic cause of ALS is an expansion of GGGGCC repeats located within the first intron of the C9ORF72 gene. Despite being located in a “non-coding” sequence, these repeats are nonetheless translated into novel and toxic proteins (Boivin et al., EMBO Journal 2020).

The candidate will investigate how these GGC and GGGGCC repeats are translated into toxic proteins using a wide range of molecular and cellular approaches (clonage, RT-qPCR, western, immunoprecipitation, immunofluorescence, transfection and cell transduction, primary cultures of mouse embryonic neurons, confocal and super resolution etc.), as well as develop novel animal models expressing these mutations through a viral strategy (AAV injection, mouse locomotor phenotyping, histology, IHC, etc.).

Overall, this proposal will help to better understand the cause of muscle and neuronal degeneration to define therapeutic strategies for these devastating diseases.

This work will take place at Institute of Genetics and Biology Molecular and Cellular (IGBMC, http://www.igbmc.fr/) a large public research laboratory comprising ~800 persons involved in 50 research groups and 12 technological platforms, including all core services essential to the present project.

Keywords:

human genetic diseases, molecular and cellular biology, mouse model, muscle, neurons.

Relevant publications:

- Boivin et al., Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders. Neuron. 2021; 109(11):1825.

- Boivin et al. C9ORF72 haploinsufficiency synergizes DPR proteins toxicity, a double hit mechanism that can be prevented by drugs activating autophagy. EMBO J. 2020; 39(4).

- Sellier et al., rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/ 2 differences. Nature Communications. 2018; 9(1):2009.

Chronic liver disease progressing to cancer such as hepatocellular carcinoma (HCC) is a major public health burden with limited therapeutic options. HCC is a leading cause of cancer-related death with rising incidence world-wide. Therapeutic discovery has been hampered by the lack of suitable models recapitulating patient liver disease and cancer for clinical translation.

Recently, our team established a 3D patient-derived liver disease model based on both cancer cell lines and patient-derived spheroids (Crouchet et al. Nature Comm 2021). Combined with scRNA-Seq and perturbation studies this model enabled us to uncover novel compounds for treatment of advanced liver disease and cancer prevention. Funded by the RHU program DELIVER of the French Research Agency (ANR), this project aims to use our established patient tissue pipeline to develop next generation patient-derived liver disease models for the investigation of liver disease and cancer biology and drug development.

Within this project, the PhD candidate will develop patient-derived spheroids and organoids combined with microfluidics, precision cut slices, aiming to build a “liver-on-a-chip model” from human patient tissues in collaboration with the Strasbourg University Hospitals. The liver-on-a-chip model will be applied to investigate the effect of therapeutic compounds using clinically relevant read outs in collaboration with Pharma. The PhD candidate will develop skills in cell and molecular biology, scRNA-Seq and related technologies, bioinformatics and build expertise in translational medicine and drug development.

KEY WORDS: Drug discovery, drug development, fibrotic liver disease, liver cancer, patient-derived models.

Relevant publications:

• Crouchet E, Li S, Sojoodi M, Bandiera S, Fujiwara N, El Saghire H,…, Hoshida Y, Fuchs BC, Baumert TF. Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation. JCI Insight. 2022 Jul 8; 7(13):e159254.
• Crouchet E, Bandiera S, Fujiwara N, Li S, El Saghire H,…, Heikenwälder M, Schuster C, Pochet N, Zeisel MB, Fuchs BC, Hoshida Y, Baumert TF. A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery. Nat Commun. 2021 Sep 17;12(1):5525.
• Jühling, F. et al. Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma. Gut 70, 157–169 (2021).

Cancers of the gastrointestinal (GI) tract are associated with a high prevalence and mortality rate worldwide. GI cancers are complex diseases, and many factors are involved in pathogenesis such as chronic inflammation, infection, and environmental and genetic factors. Current treatment options are mainly based on surgical interventions and the postoperative survival rate is still low. Moreover, despite recent advancements in molecular targeted therapies, treatment response and patient survival are still unsatisfactory. Development of complex molecular signatures including all these factors to predict disease development, complications, risk of metastasis could help to improve the development of novel treatment strategies and patient prognosis.

Previously, we have identified a pan-etiology 186-gene clinical prognostic liver signature (PLS) in diseased liver tissues robustly predicting liver disease progression and carcinogenesis in multiple patient cohorts. Based on this study, we developed a simple and robust liver cell-based system that models the PLS named cPLS for cell culture PLS. Overall, the cPLS model offers unique opportunities to discover compounds for chronic liver disease treatment and cancer prevention across the distinct liver cancer etiologies, in a fast-track high-throughput screening format (see relevant publications). 

Taking advantage of this discovery, we aim to investigate cell circuits mediating GI cancers in patient tissues to discover prognosis signatures. Thanks to a long-term collaboration with Strasbourg University Hospital, we have access to pancreatic and colon cancer patient tissues and clinical data. Within this project, the PhD candidate will analyze the whole transcriptome of patient tissues and correlate gene expression with clinical data to discover prognostic signature. These gene signatures will be used by the candidate to perform in-silico drug screening and discover novel therapeutics. In addition, the PhD student will also participate in the establishment of cell culture systems to study carcinogenesis and modulate gene signatures. The PhD student will acquire a double expertise in bio-informatic and cell and molecular biology as well as related technologies and will build expertise in translational medicine and drug discovery.

KEY WORDS: Gastrointestinal cancers, Gene signature, Drug discovery, cell culture systems.

Relevant publications:

• Crouchet E, Li S, Sojoodi M, Bandiera S, Fujiwara N, El Saghire H,…, Hoshida Y, Fuchs BC, Baumert TF. Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation. JCI Insight. 2022 Jul 8; 7(13):e159254.
• Crouchet E, Bandiera S, Fujiwara N, Li S, El Saghire H,…, Heikenwälder M, Schuster C, Pochet N, Zeisel MB, Fuchs BC, Hoshida Y, Baumert TF. A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery. Nat Commun. 2021 Sep 17;12(1):5525.
• Jühling, F. et al. Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma. Gut 70, 157–169 (2021).