bims-ginsta Biomed News
on Genome instability
Issue of 2023‒11‒19
thirty-two papers selected by
Jinrong Hu, National University of Singapore

  1. Nat Commun. 2023 Nov 16. 14(1): 7419
      Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.
  2. Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00855-9. [Epub ahead of print]83(22): 4032-4046.e6
      Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with increased cell size, we used reversible cell-cycle arrests combined with growth rate modulation to study how excessive growth affects proliferation. We find that enlarged cells upregulate p21, which limits cell-cycle progression. Cells that re-enter the cell cycle encounter replication stress that is well tolerated in physiologically sized cells but causes severe DNA damage in enlarged cells, ultimately resulting in mitotic failure and permanent cell-cycle withdrawal. We demonstrate that enlarged cells fail to recruit 53BP1 and other non-homologous end joining (NHEJ) machinery to DNA damage sites and fail to robustly initiate DNA damage-dependent p53 signaling, rendering them highly sensitive to genotoxic stress. We propose that an impaired DNA damage response primes enlarged cells for persistent replication-acquired damage, ultimately leading to cell division failure and permanent cell-cycle exit.
    Keywords:  DNA damage; cell cycle; cell growth; cell size; senescence
  3. J Cell Biol. 2024 Jan 01. pii: e202209113. [Epub ahead of print]223(1):
      Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.
  4. Stem Cell Reports. 2023 Nov 06. pii: S2213-6711(23)00417-4. [Epub ahead of print]
      Disruption of global ribosome biogenesis selectively affects craniofacial tissues with unclear mechanisms. Craniosynostosis is a congenital craniofacial disorder characterized by premature fusion of cranial suture(s) with loss of suture mesenchymal stem cells (MSCs). Here we focused on ribosomopathy disease gene Snord118, which encodes a small nucleolar RNA (snoRNA), to genetically disturb ribosome biogenesis in suture MSCs using mouse and human induced pluripotent stem cell (iPSC) models. Snord118 depletion exhibited p53 activation, increased cell death, reduced proliferation, and premature osteogenic differentiation of MSCs, leading to suture growth and craniosynostosis defects. Mechanistically, Snord118 deficiency causes translational dysregulation of ribosomal proteins and downregulation of complement pathway genes. Further complement pathway disruption by knockout of complement C3a receptor 1 (C3ar1) exacerbated MSC and suture defects in mutant mice, whereas activating the complement pathway rescued MSC cell fate and suture growth defects. Thus, ribosome biogenesis controls MSC fate via the complement pathway to prevent craniosynostosis.
    Keywords:  Snord118; complement pathway; craniosynostosis; human iPSC; ribosome biogenesis; suture mesenchymal stem cells
  5. J Cell Biol. 2024 01 01. pii: e202206013. [Epub ahead of print]223(1):
      The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.
  6. Cell Rep. 2023 Nov 14. pii: S2211-1247(23)01428-6. [Epub ahead of print]42(11): 113416
      Differentiated cardiomyocytes (CMs) must undergo diverse morphological and functional changes during postnatal development. However, the mechanisms underlying initiation and coordination of these changes remain unclear. Here, we delineate an integrated, time-ordered transcriptional network that begins with expression of genes for cell-cell connections and leads to a sequence of structural, cell-cycle, functional, and metabolic transitions in mouse postnatal hearts. Depletion of histone H2B ubiquitin ligase RNF20 disrupts this gene network and impairs CM polarization. Subsequently, assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis confirmed that RNF20 contributes to chromatin accessibility in this context. As such, RNF20 is likely to facilitate binding of transcription factors at the promoters of genes involved in cell-cell connections and actin organization, which are crucial for CM polarization and functional integration. These results suggest that CM polarization is one of the earliest events during postnatal heart development and provide insights into how RNF20 regulates CM polarity and the postnatal gene program.
    Keywords:  CP: Developmental biology; CP: Molecular biology; RNF20; cardiomyocyte polarization; cell-cell connection; chromatin accessibility; postnatal gene networks
  7. Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00861-4. [Epub ahead of print]83(22): 3946-3947
      In this issue of Molecular Cell, Crozier et al.,1 Foy et al.,2 Manohar et al.,3 and Wilson et al.4 show how excessive cell growth caused by a temporary G1 arrest leads to permanent cell cycle exit at different stages of the cell cycle.
  8. Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00853-5. [Epub ahead of print]83(22): 4062-4077.e5
      Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.
    Keywords:  DNA damage; cell cycle; cell growth; cell size; mTOR; p21(Cip1/Waf1); p38MAPK; p53; palbociclib; rapamycin
  9. Development. 2023 Nov 15. pii: dev201917. [Epub ahead of print]150(22):
      The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.
    Keywords:  Endoplasmic reticulum; Mitotic spindle; Rab proteins; Rab1; Syncytial divisions
  10. EMBO Rep. 2023 Nov 15. e57585
      Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP-binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease-dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress.
    Keywords:  DNA replication stress; DNA resection; GNL3; ORC2; origin firing
  11. Curr Opin Genet Dev. 2023 Nov 09. pii: S0959-437X(23)00113-2. [Epub ahead of print]83 102133
      The noncoding genome imparts important regulatory control over gene expression. In particular, gene enhancers represent a critical layer of control that integrates developmental and differentiation signals outside the cell into transcriptional outputs inside the cell. Recently, there has been an explosion in genomic techniques to probe enhancer control, function, and regulation. How enhancers are regulated and integrate signals in stem cell development and differentiation is largely an open question. In this review, we focus on the role gene enhancers play in muscle stem cell specification, differentiation, and progression. We pay specific attention toward the identification of muscle-specific enhancers, the binding of transcription factors to these enhancers, and how enhancers communicate to their target genes via three-dimensional looping.
  12. Dev Cell. 2023 Nov 08. pii: S1534-5807(23)00555-5. [Epub ahead of print]
      Cardiomyocytes are highly metabolic cells responsible for generating the contractile force in the heart. During fetal development and regeneration, these cells actively divide but lose their proliferative activity in adulthood. The mechanisms that coordinate their metabolism and proliferation are not fully understood. Here, we study the role of the transcription factor NFYa in developing mouse hearts. Loss of NFYa alters cardiomyocyte composition, causing a decrease in immature regenerative cells and an increase in trabecular and mature cardiomyocytes, as identified by spatial and single-cell transcriptome analyses. NFYa-deleted cardiomyocytes exhibited reduced proliferation and impaired mitochondrial metabolism, leading to cardiac growth defects and embryonic death. NFYa, interacting with cofactor SP2, activates genes linking metabolism and proliferation at the transcription level. Our study identifies a nodal role of NFYa in regulating prenatal cardiac growth and a previously unrecognized transcriptional control mechanism of heart metabolism, highlighting the importance of mitochondrial metabolism during heart development and regeneration.
    Keywords:  cardiac metabolism; cardiomyocyte proliferation; heart development; nuclear transcription factor Y
  13. Dev Cell. 2023 Nov 14. pii: S1534-5807(23)00554-3. [Epub ahead of print]
      The lung contains multiple progenitor cell types, but how their responses are choreographed during injury repair and whether this changes with age is poorly understood. We report that histone H3 lysine 9 di-methylation (H3K9me2), mediated by the methyltransferase G9a, regulates the dynamics of distal lung epithelial progenitor cells and that this regulation deteriorates with age. In aged mouse lungs, H3K9me2 loss coincided with fewer alveolar type 2 (AT2) cell progenitors and reduced alveolar regeneration but increased the frequency and activity of multipotent bronchioalveolar stem cells (BASCs) and bronchiolar progenitor club cells. H3K9me2 depletion in young mice decreased AT2 progenitor activity and impaired alveolar injury repair. Conversely, H3K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency, and accelerated bronchiolar cell injury repair. These findings indicate that during aging, the epigenetic regulation that coordinates lung progenitor cells' regenerative responses becomes dysregulated, aiding our understanding of age-related susceptibility to lung disease.
    Keywords:  EHMT2; G9a; aging; alveolar; bronchiolar; epigenetics; injury repair; lung stem cell; regeneration
  14. Nat Cell Biol. 2023 Nov 13.
      The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD+) is essential for maintaining metabolic homeostasis and preserving genomic integrity. As a co-enzyme, NAD+ plays a key role in regulating metabolic pathways, such as glycolysis and Kreb's cycle. ADP-ribosyltransferases (PARPs) and sirtuins rely on NAD+ to mediate post-translational modifications of target proteins. The activation of PARP1 in response to DNA breaks leads to rapid depletion of cellular NAD+ compromising cell viability. Therefore, the levels of NAD+ must be tightly regulated. Here we show that exogenous NAD+, but not its precursors, has a direct effect on mitochondrial activity. Short-term incubation with NAD+ boosts Kreb's cycle and the electron transport chain and enhances pyrimidine biosynthesis. Extended incubation with NAD+ results in depletion of pyrimidines, accumulation of purines, activation of the replication stress response and cell cycle arrest. Moreover, a combination of NAD+ and 5-fluorouridine selectively kills cancer cells that rely on de novo pyrimidine synthesis. We propose an integrated model of how NAD+ regulates nucleotide metabolism, with relevance to healthspan, ageing and cancer therapy.
  15. Proc Natl Acad Sci U S A. 2023 Nov 21. 120(47): e2314440120
      Ribosomal DNA (rDNA) encodes ribosomal RNA and exists as tandem repeats of hundreds of copies in the eukaryotic genome to meet the high demand of ribosome biogenesis. Tandemly repeated DNA elements are inherently unstable; thus, mechanisms must exist to maintain rDNA copy number (CN), in particular in the germline that continues through generations. A phenomenon called rDNA magnification was discovered over 50 y ago in Drosophila as a process that recovers the rDNA CN on chromosomes that harbor minimal CN. Our recent studies indicated that rDNA magnification is the mechanism to maintain rDNA CN under physiological conditions to counteract spontaneous CN loss that occurs during aging. Our previous studies that explored the mechanism of rDNA magnification implied that asymmetric division of germline stem cells (GSCs) may be particularly suited to achieve rDNA magnification. However, it remains elusive whether GSCs are the unique cell type that undergoes rDNA magnification or differentiating germ cells are also capable of magnification. In this study, we provide empirical evidence that suggests that rDNA magnification operates uniquely in GSCs, but not in differentiating germ cells. We further provide computer simulation that suggests that rDNA magnification is only achievable through asymmetric GSC divisions. We propose that despite known plasticity and transcriptomic similarity between GSCs and differentiating germ cells, GSCs' unique ability to divide asymmetrically serves a critical role of maintaining rDNA CN through generations, supporting germline immortality.
    Keywords:  Drosophila germline; germline immortality; rDNA copy number maintenance; ribosomal DNA
  16. Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00857-2. [Epub ahead of print]83(22): 4047-4061.e6
      CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.
    Keywords:  CDK4/6; breast cancer; cell cycle; cell growth; chemotherapy; growth factors; oncogenes; p21; p53; replication stress
  17. bioRxiv. 2023 Oct 24. pii: 2023.07.22.550178. [Epub ahead of print]
      The interchromatin space in the cell nucleus contains various membrane-less nuclear bodies. Recent findings indicate that nuclear speckles, comprising a distinct nuclear body, exhibit interactions with certain chromatin regions in a ground state. Key questions are how this ground state of chromatin-nuclear speckle association is established and what are the gene regulatory roles of this layer of nuclear organization. We report here that chromatin structural factors CTCF and cohesin are required for full ground state association between DNA and nuclear speckles. Disruption of ground state DNA-speckle contacts via either CTCF depletion or cohesin depletion had minor effects on basal level expression of speckle-associated genes, however we show strong negative effects on stimulus-dependent induction of speckle-associated genes. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association. In contrast to reduction of CTCF or RAD21, depletion of the cohesin releasing factor WAPL stabilized cohesin on chromatin and DNA-speckle contacts, resulting in enhanced inducibility of speckle-associated genes. In addition, we observed disruption of chromatin-nuclear speckle association in patient derived cells with Cornelia de Lange syndrome (CdLS), a congenital neurodevelopmental diagnosis involving defective cohesin pathways, thus revealing nuclear speckles as an avenue for therapeutic inquiry. In summary, our findings reveal a mechanism to establish the ground organizational state of chromatin-speckle association, to promote gene inducibility, and with relevance to human disease.
  18. Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00854-7. [Epub ahead of print]83(22): 4078-4092.e6
      Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.
    Keywords:  CDK inhibition; CDK7 inhibitor; cancer treatment; cell cycle; cell size; cell-cycle arrest; cellular growth; mTOR singaling; proliferation; samuraciclib; senescence
  19. Dev Cell. 2023 Nov 07. pii: S1534-5807(23)00553-1. [Epub ahead of print]
      During meiosis, the chromatin and transcriptome undergo prominent switches. Although recent studies have explored the genome reorganization during spermatogenesis, the chromatin remodeling in oogenesis and characteristics of homologous pairing remain largely elusive. We comprehensively compared chromatin structures and transcriptomes at successive substages of meiotic prophase in both female and male mice using low-input high-through chromosome conformation capture (Hi-C) and RNA sequencing (RNA-seq). Compartments and topologically associating domains (TADs) gradually disappeared and slowly recovered in both sexes. We found that homologs adopted different sex-conserved pairing strategies prior to and after the leptotene-to-zygotene transition, changing from long interspersed nuclear element (LINE)-enriched compartments B to short interspersed nuclear element (SINE)-enriched compartments A. We complemented marker genes and predicted the sex-specific meiotic sterile genes for each substage. This study provides valuable insights into the similarities and distinctions between sexes in chromosome architecture, homologous pairing, and transcriptome during meiotic prophase of both oogenesis and spermatogenesis.
    Keywords:  3D genome; homologous pairing; meiotic prophase; sexual dimorphism; transcriptome
  20. Cell Rep. 2023 Nov 13. pii: S2211-1247(23)01424-9. [Epub ahead of print]42(11): 113412
      RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.
    Keywords:  53BP1; CP: Molecular biology; DNA replication; Okazaki fragments; RNA-binding protein
  21. Science. 2023 Nov 17. 382(6672): eadg3053
      Cells remember their identities, in part, by using epigenetic marks-chemical modifications placed along the genome. How can mark patterns remain stable over cell generations despite their constant erosion by replication and other processes? We developed a theoretical model that reveals that three-dimensional (3D) genome organization can stabilize epigenetic memory as long as (i) there is a large density difference between chromatin compartments, (ii) modifying "reader-writer" enzymes spread marks in three dimensions, and (iii) the enzymes are limited in abundance relative to their histone substrates. Analogous to an associative memory that encodes memory in neuronal connectivity, mark patterns are encoded in a 3D network of chromosomal contacts. Our model provides a unified account of diverse observations and reveals a key role of 3D genome organization in epigenetic memory.
  22. Cell Rep. 2023 Nov 10. pii: S2211-1247(23)01440-7. [Epub ahead of print]42(11): 113428
      H3K9 methylation (H3K9me) marks transcriptionally silent genomic regions called heterochromatin. HP1 proteins are required to establish and maintain heterochromatin. HP1 proteins bind to H3K9me, recruit factors that promote heterochromatin formation, and oligomerize to form phase-separated condensates. We do not understand how these different HP1 properties are involved in establishing and maintaining transcriptional silencing. Here, we demonstrate that the S. pombe HP1 homolog, Swi6, can be completely bypassed to establish silencing at ectopic and endogenous loci when an H3K4 methyltransferase, Set1, and an H3K14 acetyltransferase, Mst2, are deleted. Deleting Set1 and Mst2 enhances Clr4 enzymatic activity, leading to higher H3K9me levels and spreading. In contrast, Swi6 and its capacity to oligomerize were indispensable during epigenetic maintenance. Our results demonstrate the role of HP1 proteins in regulating histone modification crosstalk during establishment and identify a genetically separable function in maintaining epigenetic memory.
    Keywords:  CP: Molecular biology; H3K9 methylation; chromatin compaction; condensates; epigenetics; euchromatin; heterochromatin; histones; inheritance; silencing; yeast
  23. bioRxiv. 2023 Oct 23. pii: 2023.10.23.563604. [Epub ahead of print]
      Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.
  24. Nature. 2023 Nov 15.
      Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.
  25. Mol Cell. 2023 Nov 02. pii: S1097-2765(23)00848-1. [Epub ahead of print]
      Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
    Keywords:  K6-linked ubiquitylation; RNA-protein crosslinks; RNF14; VCP; quantitative proteomics; reactive aldehydes; ribosome; translation
  26. Cell Rep. 2023 Nov 16. pii: S2211-1247(23)01452-3. [Epub ahead of print]42(11): 113440
      Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.
    Keywords:  AMPA receptor desensitization; CP: Neuroscience; Developmental biology; aII amacrine cell; excitatory postsynaptic response; night vision; quantal analysis; retinal circuits; ribbon synapse; rod bipolar cell; synaptic transmission
  27. bioRxiv. 2023 Oct 23. pii: 2023.10.20.563147. [Epub ahead of print]
      Aging is the greatest risk factor for breast cancer; however, how age-related cellular and molecular events impact cancer initiation is unknown. We investigate how aging rewires transcriptomic and epigenomic programs of mouse mammary glands at single cell resolution, yielding a comprehensive resource for aging and cancer biology. Aged epithelial cells exhibit epigenetic and transcriptional changes in metabolic, pro-inflammatory, or cancer-associated genes. Aged stromal cells downregulate fibroblast marker genes and upregulate markers of senescence and cancer-associated fibroblasts. Among immune cells, distinct T cell subsets ( Gzmk + , memory CD4 + , γδ) and M2-like macrophages expand with age. Spatial transcriptomics reveal co-localization of aged immune and epithelial cells in situ . Lastly, transcriptional signatures of aging mammary cells are found in human breast tumors, suggesting mechanistic links between aging and cancer. Together, these data uncover that epithelial, immune, and stromal cells shift in proportions and cell identity, potentially impacting cell plasticity, aged microenvironment, and neoplasia risk.
  28. Nat Struct Mol Biol. 2023 Nov;30(11): 1816-1825
      A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. Start codon-associated ribosomal frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra that are unannotated in the human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational 'noise' in nutrient stress adaptation.
  29. bioRxiv. 2023 Nov 02. pii: 2023.10.31.564750. [Epub ahead of print]
      The mammalian mitochondrial genome encodes thirteen oxidative phosphorylation system proteins, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native human mitochondrial mt-mRNA structurome. We gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique programmed ribosomal frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts.
  30. Nat Commun. 2023 Nov 11. 14(1): 7308
      RIG-I is an essential innate immune receptor that responds to infection by RNA viruses. The RIG-I signaling cascade is mediated by a series of post-translational modifications, the most important of which is ubiquitination of the RIG-I Caspase Recruitment Domains (CARDs) by E3 ligase Riplet. This is required for interaction between RIG-I and its downstream adapter protein MAVS, but the mechanism of action remains unclear. Here we show that Riplet is required for RIG-I signaling in the presence of both short and long dsRNAs, establishing that Riplet activation does not depend upon RIG-I filament formation on long dsRNAs. Likewise, quantitative Riplet-RIG-I affinity measurements establish that Riplet interacts with RIG-I regardless of whether the receptor is bound to RNA. To understand this, we solved high-resolution cryo-EM structures of RIG-I/RNA/Riplet complexes, revealing molecular interfaces that control Riplet-mediated activation and enabling the formulation of a unified model for the role of Riplet in signaling.
  31. Nat Commun. 2023 Nov 17. 14(1): 7445
      Hydraulic fracturing plays a major role in cavity formation during embryonic development, when pressurized fluid opens microlumens at cell-cell contacts, which evolve to form a single large lumen. However, the fundamental physical mechanisms behind these processes remain masked by the complexity and specificity of biological systems. Here, we show that adhered lipid vesicles subjected to osmotic stress form hydraulic microlumens similar to those in cells. Combining vesicle experiments with theoretical modelling and numerical simulations, we provide a physical framework for the hydraulic reconfiguration of cell-cell adhesions. We map the conditions for microlumen formation from a pristine adhesion, the emerging dynamical patterns and their subsequent maturation. We demonstrate control of the fracturing process depending on the applied pressure gradients and the type and density of membrane bonds. Our experiments further reveal an unexpected, passive transition of microlumens to closed buds that suggests a physical route to adhesion remodeling by endocytosis.
  32. bioRxiv. 2023 Nov 04. pii: 2023.11.02.565392. [Epub ahead of print]
      Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates the G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the SBF complex that promotes transcription of the cyclins CLN1 and CLN2 . In late-G1, Whi5 is rapidly hyper-phosphorylated by Cln1,2 in complex with the cyclin-dependent kinase Cdk1. This hyper-phosphorylation inactivates Whi5 and excludes it from the nucleus. Here, we set out to determine the molecular mechanisms responsible for Whi5's multi-site phosphorylation and how they regulate the cell cycle. To do this, we first identified the 19 Whi5 sites that are appreciably phosphorylated and then determined which of these sites are responsible for G1 hypo-phosphorylation. Mutation of 7 sites removed G1 hypo-phosphorylation, increased cell size, and delayed the G1/S transition. Moreover, the rapidity of Whi5 hyper-phosphorylation in late G1 depends on 'priming' sites that dock the Cks1 subunit of Cln1,2-Cdk1 complexes. Hyper-phosphorylation is crucial for Whi5 nuclear export, normal cell size, full expression of SBF target genes, and timely progression through both the G1/S transition and S/G2/M phases. Thus, our work shows how Whi5 phosphorylation regulates the G1/S transition and how it is required for timely progression through S/G2/M phases and not only G1 as previously thought.