NOMIS Researcher(s)
Published in Molecular Cell
FUS is an RNA binding protein associated with several neurodegenerative diseases, for which mode of nucleic acid binding has been elusive. Loughlin et al. solved the solution structure of FUS bound to RNA, revealing a sequence-specific recognition for a GGU motif and an unusual shape recognition of a stem loop by two separate domains.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Journal of Cell Biology
Many adult tissues contain postmitotic cells as old as the host organism. Te only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specifc enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our fndings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Current Biology
One of the features that distinguishes modern humans from our extinct relatives and ancestors is a globular shape of the braincase [1–4]. As the endocranium closely mirrors the outer shape of the brain, these differences might reflect altered neural architecture [4, 5]. However, in the absence of fossil brain tissue, the underlying neuroanatomical changes as well as their genetic bases remain elusive. To better understand the biological foundations of modern human endocranial shape, we turn to our closest extinct relatives: the Neandertals. Interbreeding between modern humans and Neandertals has resulted in introgressed fragments of Neandertal DNA in the genomes of present-day non-Africans [6, 7]. Based on shape analyses of fossil skull endocasts, we derive a measure of endocranial globularity from structural MRI scans of thousands of modern humans and study the effects of introgressed fragments of Neandertal DNA on this phenotype. We find that Neandertal alleles on chromosomes 1 and 18 are associated with reduced endocranial globularity. These alleles influence expression of two nearby genes, UBR4 and PHLPP1, which are involved in neurogenesis and myelination, respectively. Our findings show how integration of fossil skull data with archaic genomics and neuroimaging can suggest developmental mechanisms that may contribute to the unique modern human endocranial shape.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Nature Reviews Genetics
The genome is packaged and organized nonrandomly within the 3D space of the nucleus to promote efficient gene expression and to faithfully maintain silencing of heterochromatin. The genome is enclosed within the nucleus by the nuclear envelope membrane, which contains a set of proteins that actively participate in chromatin organization and gene regulation. Technological advances are providing views of genome organization at unprecedented resolution and are beginning to reveal the ways that cells co-opt the structures of the nuclear periphery for nuclear organization and gene regulation. These genome regulatory roles of proteins of the nuclear periphery have important influences on development, disease and ageing.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Life Science Alliance
FUS (fused in sarcoma) plays a key role in several steps of RNA metabolism, and dominant mutations in this protein are associated with neurodegenerative diseases. Here, we show that FUS is a component of the cellular response to topoisomerase I (TOP1)–induced DNA breakage; relocalising to the nucleolus in response to RNA polymerase II (Pol II) stalling at sites of TOP1-induced DNA breaks. This relocalisation is rapid and dynamic, reversing following the removal of TOP1-induced breaks and coinciding with the recovery of global transcription. Importantly, FUS relocalisation following TOP1-induced DNA breakage is associated with increased FUS binding at sites of RNA polymerase I transcription in ribosomal DNA and reduced FUS binding at sites of RNA Pol II transcription, suggesting that FUS relocates from sites of stalled RNA Pol II either to regulate pre-mRNA processing during transcriptional stress or to modulate ribosomal RNA biogenesis. Importantly, FUS-mutant patient fibroblasts are hypersensitive to TOP1-induced DNA breakage, highlighting the possible relevance of these findings to neurodegeneration.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Nature Communications
A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the “CRISPY” mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes or of gene encoding a blue fluorescent protein in 27% of cells, to our knowledge the highest editing efficiency in human induced pluripotent stem cells described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.3- to 4.0-fold, allowing almost 20% of chromosomes to be edited. The components of the CRISPY mix do not always increase the editing efficiency in the immortalized or primary cell lines tested, suggesting that employed repair pathways are cell-type specific.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Nature Communications
Genetic, epigenetic, and environmental factors contribute to the multifactorial disorder progressive supranuclear palsy (PSP). Here, we study epigenetic changes by genome-wide analysis of DNA from postmortem tissue of forebrains of patients and controls and detect significant (P < 0.05) methylation differences at 717 CpG sites in PSP vs. controls. Four-hundred fifty-one of these sites are associated with protein-coding genes. While differential methylation only affects a few sites in most genes, DLX1 is hypermethylated at multiple sites. Expression of an antisense transcript of DLX1, DLX1AS, is reduced in PSP brains. The amount of DLX1 protein is increased in gray matter of PSP forebrains. Pathway analysis suggests that DLX1 influences MAPT-encoded Tau protein. In a cell system, overexpression of DLX1 results in downregulation of MAPT while overexpression of DLX1AS causes upregulation of MAPT. Our observations suggest that altered DLX1 methylation and expression contribute to pathogenesis of PSP by influencing MAPT.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Nature
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3′-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Cell
Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism. Defense strategies that are cooperative rather than antagonistic can promote asymptomatic infection and select for reduced virulence in a bacterial pathogen.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Cell Reports
The primarily nuclear RNA-binding protein FUS (fused in sarcoma) forms pathological cytoplasmic inclusions in a subset of early-onset amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. In response to cellular stress, FUS is recruited to cytoplasmic stress granules, which are hypothesized to act as precursors of pathological inclusions. We monitored the stress-induced nucleocytoplasmic shuttling of endogenous FUS in an ex vivo mouse CNS model and human neural networks. We found that hyperosmolar, but not oxidative, stress induced robust cytoplasmic translocation of neuronal FUS, with transient nuclear clearance and loss of function. Surprisingly, this reaction is independent of stress granule formation and the molecular pathways activated by hyperosmolarity. Instead, it represents a mechanism mediated by cytoplasmic redistribution of Transportin 1/2 and is potentiated by transcriptional inhibition. Importantly, astrocytes, which remain unaffected in ALS/FTD-FUS, are spared from this stress reaction that may signify the initial event in the development of FUS pathology.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in EMBO Molecular Medicine
CHCHD10 mutations are linked to amyotrophic lateral sclerosis, but their mode of action is unclear. In a 29-year-old patient with rapid disease progression, we discovered a novel mutation (Q108P) in a conserved residue within the coiled-coil-helix-coiled-coil-helix (CHCH) domain. The aggressive clinical phenotype prompted us to probe its pathogenicity. Unlike the wild-type protein, mitochondrial import of CHCHD10 Q108P was blocked nearly completely resulting in diffuse cytoplasmic localization and reduced stability. Other CHCHD10 variants reported in patients showed impaired mitochondrial import (C122R) or clustering within mitochondria (especially G66V and E127K) often associated with reduced expression. Truncation experiments suggest mitochondrial import of CHCHD10 is mediated by the CHCH domain rather than the proposed N-terminal mitochondrial targeting signal. Knockdown of Mia40, which introduces disulfide bonds into CHCH domain proteins, blocked mitochondrial import of CHCHD10. Overexpression of Mia40 rescued mitochondrial import of CHCHD10 Q108P by enhancing disulfide-bond formation. Since reduction in CHCHD10 inhibits respiration, mutations in its CHCH domain may cause aggressive disease by impairing mitochondrial import. Our data suggest Mia40 upregulation as a potential therapeutic salvage pathway.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Cell
Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import—namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration. Phase separation of FUS is regulated by both arginine methylation and binding to the nuclear import receptor Transportin/Karyopherin-β2, and this control can be compromised in the neurodegenerative diseases FTD and ALS.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Aging Cell
Aging is a major driving force underlying dementia, such as that caused by Alzheimer’s disease (AD). While the idea of targeting aging as a therapeutic strategy is not new, it remains unclear how closely aging and age-associated diseases are coupled at the molecular level. Here, we discover a novel molecular link between aging and dementia through the identification of the molecular target for the AD drug candidate J147. J147 was developed using a series of phenotypic screening assays mimicking disease toxicities associated with the aging brain. We have previously demonstrated the therapeutic efficacy of J147 in several mouse models of AD. Here, we identify the mitochondrial α-F 1 -ATP synthase (ATP5A) as a target for J147. By targeting ATP synthase, J147 causes an increase in intracellular calcium leading to sustained calcium/calmodulin-dependent protein kinase kinase β (CAMKK2)-dependent activation of the AMPK/mTOR pathway, a canonical longevity mechanism. Accordingly, modulation of mitochondrial processes by J147 prevents age-associated drift of the hippocampal transcriptome and plasma metabolome in mice and extends lifespan in drosophila. Our results link aging and age-associated dementia through ATP synthase, a molecular drug target that can potentially be exploited for the suppression of both. These findings demonstrate that novel screens for new AD drug candidates identify compounds that act on established aging pathways, suggesting an unexpectedly close molecular relationship between the two.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in RNA
The term “nonsense-mediated mRNA decay” (NMD) originally described the degradation of mRNAs with premature translation–termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%–10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5–SMG7 or SMG5–PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5??–3?? exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Cell Stress
Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5′ and 3′ ends and are recognized by a separate pre-mRNA processing machinery denoted as the minor spliceosome. This spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on minor spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Stem Cell Reports
Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG) phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Cell
Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes. Neuronal poly-GA aggregates linked to amyotrophic lateral sclerosis and frontotemporal dementia selectively sequester proteasomes.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Molecular Biology of the Cell
CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to ?50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Life Science Alliance
Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Over-expressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule–like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Genes and Development
The total number of nuclear pore complexes (NPCs) per nucleus varies greatly between different cell types and is known to change during cell differentiation and cell transformation. However, the underlying mechanisms that control how many nuclear transport channels are assembled into a given nuclear envelope remain unclear. Here, we report that depletion of the NPC basket protein Tpr, but not Nup153, dramatically increases the total NPC number in various cell types. This negative regulation of Tpr occurs via a phosphorylation cascade of extracellular signal-regulated kinase (ERK), the central kinase of the mitogen-activated protein kinase (MAPK) pathway. Tpr serves as a scaffold for ERK to phosphorylate the nucleoporin (Nup) Nup153, which is critical for early stages of NPC biogenesis. Our results reveal a critical role of the Nup Tpr in coordinating signal transduction pathways during cell proliferation and the dynamic organization of the nucleus.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
NOMIS Researcher(s)
Published in Nature Communications
Premature aging disorders provide an opportunity to study the mechanisms that drive aging. In Hutchinson-Gilford progeria syndrome (HGPS), a mutant form of the nuclear scaffold protein lamin A distorts nuclei and sequesters nuclear proteins. We sought to investigate protein homeostasis in this disease. Here, we report a widespread increase in protein turnover in HGPS-derived cells compared to normal cells. We determine that global protein synthesis is elevated as a consequence of activated nucleoli and enhanced ribosome biogenesis in HGPS-derived fibroblasts. Depleting normal lamin A or inducing mutant lamin A expression are each sufficient to drive nucleolar expansion. We further show that nucleolar size correlates with donor age in primary fibroblasts derived from healthy individuals and that ribosomal RNA production increases with age, indicating that nucleolar size and activity can serve as aging biomarkers. While limiting ribosome biogenesis extends lifespan in several systems, we show that increased ribosome biogenesis and activity are a hallmark of premature aging.
Research field(s)
Health Sciences, Biomedical Research, Developmental Biology
