Biology subjects

Lafontaine, D. L.

Publications and source records attributed to Lafontaine, D. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Stepwise reorganization of chromosome conformation and nuclear organization during stem cell differentiation

Nuclear organization is a fundamental feature of cell identity and cell fate determination. Compared with differentiated cells, pluripotent stem cells exhibit markedly distinct nuclear architecture at multiple levels, including chromosome folding, lamina association, histone modification landscapes and nuclear body organization. Although individual aspects of this reorganization have been characterized, how and in what order these features are remodeled as cells commit to a lineage remains poorly understood. Here, we temporally map nuclear reorganization across five stages of an in vitro differentiation system of human embryonic stem cells through definitive endoderm and hepatoblast intermediates into hepatocyte-like cells. By integrating Hi-C, RNA-seq, ATAC-seq, ChIP-seq, and CUT&RUN data, we establish a genome-wide framework linking structural, epigenetic, and transcriptional changes across differentiation. Combined with immunofluorescence imaging, chromosome painting, and liquid chromatin Hi-C (LC-Hi-C) we show that nuclear reorganization proceeds in a stepwise and temporally ordered manner through three major transitions. In the first transition, chromosomes condense into defined territories concurrent with transient anchoring of centromere-proximal regions to the nuclear periphery. In a second transition, active and inactive chromatin segregate, leading to stronger compartmentalization. This coincides with deposition and peripheralization of H3K9me2-marked chromatin and morphological changes in nuclear speckles, while chromatin conformation at speckle-associated regions stabilizes. In a third transition, after lineage-specific genes are activated, chromatin interactions globally stabilize, establishing a more stable nuclear architecture on top of these earlier large-scale structural rearrangements. Together, our results define a stepwise framework for nuclear reorganization during human embryonic stem cell differentiation and reveal that the transition from pluripotency to a differentiated state proceeds through coordinated, temporally ordered structural events.

genomics↗

bioRxiv2026

Formation of an RNA-mediated nuclear compartment

Mammalian nuclei are spatially compartmentalized so that active and inactive segments of the genome occupy separate sub-nuclear neighborhoods. Compartments are often found in association with nuclear structures such as the nuclear lamina, nucleoli, and nuclear speckles (speckles), suggesting links between them. The molecular mechanisms by which compartments form remain largely unknown. Speckles are nuclear bodies that contain high concentrations of RNA splicing factors and associated chromatin has a high density of highly expressed genes. Combining liquid chromatin Hi-C to quantify chromatin interaction lifetimes genome-wide, immunofluorescence, fluorescence in situ hybridization, live cell imaging, and nascent transcript analysis, we have determined the biophysical and molecular basis of the speckle-associated chromosomal compartment. We find that genomic regions making up this compartment are stably glued together. Surprisingly, removal of speckles by rapid depletion of SON and SRRM2 that form the structural scaffold of these bodies shows that this stable association is not dependent on the speckle itself. Instead, we find that RNA molecules are the molecular glue that forms the biophysical basis of the speckle chromatin compartment. Based on observations that promoters and enhancers are also engaged in stable long-lived chromatin interactions that are independent of RNA, we propose a pathway for formation of the speckle chromosomal compartment: Initial stable clustering of promoters and enhancers is followed by production of (nascent) RNA. The exceptionally high density of GC-rich RNA emerging from speckle-associated loci forms a glue that holds these loci together and facilitates recruitment of speckle components. The result is a structurally stable nuclear compartment that facilitates efficient splicing.

genomics↗

bioRxiv2025

Intrinsically Disordered Regions Form Nucleoli and Cajal Bodies While Fostering RNA Modification

One of the densest compartments in the cell is the dense fibrillar component (DFC) of the nucleolus, consisting mainly of nascent ribosomal RNA (rRNA), small nucleolar ribonucleoproteins (snoRNPs) and their chaperone Nopp140. How this biomolecular condensate is formed and what underlies its structure is poorly understood like that of most liquid-liquid phase separated condensates. Although we established that Nopp140 is important for the cohesiveness of the DFC and for rRNA modification, it is not known how this is achieved. Here we demonstrate that Nopp140 concentrates intrinsically disordered and nuclear localization signal (NLS)-rich protein regions (IDRs), including a newly identified RNA polymerase I C-terminal domain (CTD) of the RNA polymerase I associated factor PAF49. Altogether, this network forms the DFC, a liquid-liquid phase separated biomolecular condensate that fosters rRNA modification. This mechanism ensures near 100 percent modification efficiency at some 200 nucleotides in every one of the 10 million or so rRNAs per cell.

cell biology↗

bioRxiv2025

Zα domain-dependent ZBP1 condensate formation induces an amyloidal necroptotic signalling complex

ZBP1 restricts viral replication by inducing host cell death. ZBP1 recognises Z-RNA or Z-DNA, left-handed double-stranded RNA or DNA structures that accumulate after virus infection. How the interaction between Z-RNA/DNA and ZBP1 governs its activation and how this mediates downstream signalling remains unclear. Using herpes simplex virus 1 (HSV-1) as an activator of human ZBP1 we find that binding of the N-terminal Z domains of ZBP1 to Z-RNA induces ZBP1 condensate formation. This then mediates oligomerisation of the RIP homotypic interaction motifs (RHIMs) of ZBP1 establishing an amyloidal signalling complex with RIPK1 and RIPK3 that induces necroptotic cell death. We find that the kinase activity of RIPK1 is essential for RIPK1 and RIPK3 oligomerisation downstream of human ZBP1. Finally, the HSV-1-encoded RHIM-containing protein ICP6, does not interfere with Z domain-mediated ZBP1 condensate formation, but instead prevents downstream RIPK1 and RIPK3 oligomerisation thereby inhibiting necroptosis and promoting viral growth. Together, this shows that ZBP1 condensate formation restricts HSV-1 infection by promoting host cell necroptosis.

immunology↗

bioRxiv2025

AquIRE reveals multiple mechanisms of clinically induced RNA damage and the conservation and dynamics of glycoRNAs

RNA is subject to many modifications, from small chemical changes such as methylation through to conjugation of biomolecules such as glycans. As well as these endogenously written modifications, RNA is also exposed to damage induced by its environment. Certain clinical compounds are known to drive covalent modifications of RNA with a growing appreciation for how these affect function. To understand the regulation of these modifications we need a reliable, sensitive and rapid methodology for their quantification. Thus, we developed AquIRE and applied it to the analysis of drug-induced RNA damage, showing this to be widespread with intricate temporal dynamics. Using the same methodology we identify RNA:protein crosslinking and the rewriting of the epitranscriptome as a consequence of clinical RNA damage. We also demonstrate how liquid-liquid phase separation increases RNA damage and expand the horizons of the glycoRNA world across the kingdoms of life and into cell-free glycoRNA.

molecular biology↗

bioRxiv2025

Redefining human pre-rRNA processing at single nucleotide resolution using long read Nanopore sequencing

Ribosome biogenesis requires the synthesis and processing of precursor rRNAs (pre-rRNAs) into mature rRNAs. Traditional methods like northern blotting and metabolic labeling offer limited resolution. We present NanoRibolyzer, a nanopore-based, long-read sequencing approach that enables ab initio identification and quantification of rRNA precursors. Using both supervised and unsupervised mapping, it detects known and novel pre-rRNA species and defines cleavage sites at single-nucleotide resolution. A simple cell fractionation protocol provides spatial separation of nuclear and cytoplasmic pre-rRNAs. Targeted knockdowns quantify intermediate accumulations, revealing condition-specific processing fingerprints with biomarker potential. Pseudouridine mapping shows that the primary 47S transcript is extensively modified, while aberrant products are not. With its high resolution and unique mapping strategy, NanoRibolyzer offers new insights into rRNA processing and modification, enhancing our understanding of ribosome biogenesis.

cell biology↗

bioRxiv2024

Epitranscriptomic rRNA fingerprinting reveals tissue-of-origin and tumor-specific signatures

Mammalian ribosomal RNA (rRNA) molecules are highly abundant RNAs, decorated with over 220 rRNA modifications. Previous works have shown that some rRNA modification types can be dynamically regulated; however, how and when the mammalian rRNA modification landscape is remodeled remains largely unexplored. Here, we employ direct RNA nanopore sequencing to chart the human and mouse rRNA epitranscriptome across tissues, developmental stages, cell types and disease. Our analyses reveal multiple rRNA sites that are differentially modified in a tissue- and/or developmental stage-specific manner, including previously unannotated modified sites. We demonstrate that rRNA modification patterns can be used for tissue and cell type identification, which we hereby term epitranscriptomic fingerprinting. We then explore rRNA modification patterns in normal-tumor matched samples from lung cancer patients, finding that epitranscriptomic fingerprinting accurately classifies clinical samples into normal and tumor groups from only 250 reads per sample, demonstrating the potential of rRNA modifications as diagnostic biomarkers.

molecular biology↗

bioRxiv2024

Mapping and engineering RNA-controlled architecture of the multiphase nucleolus

Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a layered multiphase liquid-like structure and the site of ribosome biogenesis. In the nucleolus, ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps that ultimately result in formation of the ribosomal small subunit (SSU) and large subunit (LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. Furthermore, by generating synthetic de novo nucleoli through an engineered rDNA plasmid system in cells, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.

cell biology↗

bioRxiv2023

Detecting Material State Changes in the Nucleolus by Label-free Digital Holographic Microscopy

Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects its function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension cells. Subtle nucleolar alterations induced by drug treatment or by depletion of ribosomal proteins were efficiently detected by DHM. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images of cultured human cells (HeLa). Holograms containing cell optical thickness information allowed us to define a novel nucleolar index which we used to distinguish nucleoli whose material state had been optogenetically modulated. We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli, including material state, without any staining.

cell biology↗

bioRxiv2022

Chromosome decompaction and cohesin direct Topoisomerase II activity to establish and maintain an unentangled interphase genome

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations and multi-contact 3C, and propose that, in contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a Topoisomerase II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias Topoisomerase II activity towards decatenation. At the second stage, the loops are released, and formation of new entanglements is prevented by lower Topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed, in experiment and models, a normal interphase state cannot be acquired.

genomics↗

bioRxiv2021

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution

NAT10 is an essential enzyme that catalyzes the formation of N4-acetylcytidine (ac4C) in eukaryotic transfer RNA (tRNA) and 18S ribosomal RNA (rRNA). Recent studies in human cells suggested that rRNA acetylation is dependent on SNORD13, a non-canonical box C/D small nucleolar RNA (SNORD) predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10s essential function in pre-rRNA processing remain to be defined. Here, we used CRISPR-Cas9 genome editing to formally demonstrate that SNORD13 is required for acetylation of a single cytidine residue of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for yeast or human cell growth, ribosome biogenesis, translation, and the development of multicellular metazoan model organisms. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation machinery that led to the characterization of many novel SNORD13 genes in phylogenetically-distant metazoans and more deeply rooted photosynthetic organisms. This includes an atypical SNORD13-like RNA in D. melanogaster which appears to guide ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that C. elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Altogether, our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across the eukaryotic tree of life and raise new questions regarding the biological function and evolutionary persistence of this highly conserved rRNA base modification.

molecular biology↗

bioRxiv2021

Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration. AUTHOR SUMMARYRibosomes are essential nanomachines responsible for protein production in all cells. Ribosomopathies are diseases caused by improper ribosome formation due to mutations in ribosomal proteins or ribosome assembly factors. Such diseases primarily affect the brain and blood, and it is unclear how malfunctioning of a process as general as ribosome formation can lead to tissue-specific diseases. Here we have examined how fibrillarin, an enzyme which modifies ribosomal RNA by adding methyl groups at specific sites, affects early embryonic development in the frog Xenopus laevis. We have revealed its importance in the maturation of cells forming an embryonic structure called the neural crest. Fibrillarin depletion leads to reduced eye size and abnormal head shape, reminiscent of other conditions such as Treacher Collins syndrome. Molecularly, the observed phenotypes are explainable by increased p53-dependent programmed cell death triggered by inhibition of certain pre-rRNA processing steps. Our systematic investigation of the ribosomal RNA 2-O methylation repertoire across development has further revealed hypomodification at a late stage of development, which might play a role in late developmental transitions involving differential translation by compositionally different ribosomes.

developmental biology↗

bioRxiv2021

Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridines, 2-O-methylation of riboses and acetylation of cytidines. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

molecular biology↗

bioRxiv2021

Systematic mapping of small nucleolar RNA targets in human cells

Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

cell biology↗

bioRxiv2021

Nucleolar stress controls mutant Huntingtin toxicity and monitors Huntington disease progression

Transcriptional and cellular stress surveillance deficits are hallmarks of Huntingtons disease (HD), a fatal autosomal dominant neurodegenerative disorder, caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD remain unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo is elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases mHTT disperse state in the nucleus, exacerbating the motor deficits. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its nucleolar localization. NPM1 de-localization occurs in the striatum and in the skeletal muscle of the progressive zQ175 knock-in HD mouse model, mimicking the phenotype of HD patients in skeletal muscle biopsies. Taken together, we showed that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identified NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.

neuroscience↗

bioRxiv2021

Nopp140-chaperoned 2'-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

cell biology↗

bioRxiv2020

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved locomotion at midlife and resistance against heat stress, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodelled leading to reduced production of collagens, loss of structural integrity of the cuticle and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal physiology.

cell biology↗

bioRxiv2020

Mechanism of monoallelic expression and allelic rheostat role of DNA methylation

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

genomics↗