Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using ...mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A—a membrane-bound protein interacting with nucleoids—as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.
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•mtDSBs lead to severe mitochondrial defects and impaired protein import•mtDSBs trigger an integrated stress response (ISR)•Suppressing ISR amplifies mitochondrial defects and decelerates mtDNA recovery•ATAD3A potentially signals from damaged genomes to the inner mitochondrial membrane
Fu et al. show that mitochondrial DNA double-strand breaks (mtDSBs) severely impair mitochondrial function, affecting mitochondrial protein import and membrane potential. Such damage initiates an integrated stress response (ISR) critical for mitochondrial stability. The study highlights ATAD3A as key in signaling between damaged genomes and the mitochondrial membrane.
Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. ...Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.
Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. ...Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.
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•Enhancers have a minor role in regulating telomerase during development•Alternative splicing of exon 2 is a major determinant of hTERT levels•Exon 2 skipping triggers hTERT mRNA decay in somatic cells•SON regulates hTERT splicing, and its mutation manifests in telomerase insufficiency
Human telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere length. Penev et al. identify a critical role for hTERT alternative splicing in ensuring robust telomerase activation in pluripotent cells and its repression in somatic cells.
DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure ...fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses ...that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly ...understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.
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•Live-cell and single-molecule imaging of telomerase RNA by MS2 tagging•Telomerase reverse transcriptase drives hTR exit from Cajal bodies•ATM (and ATR) facilitates hTR recruitment to telomeres•RNA-DNA base pairing promotes telomerase retention at telomeres
Telomerase is the means by which many cancer cells achieve immortality. Laprade et al. developed an MS2-tagging approach to visualize single particles of hTR in cancer cells and reveal telomerase dynamics as hTR transits through Cajal bodies and is recruited and retained at telomeres.
Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and ...accelerated tumor development. Here, we define the impact of mutant
alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with
mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
Telomeres protect chromosome ends through the interaction of telomeric repeats with shelterin, a protein complex that represses DNA damage signaling and DNA repair reactions. The telomeric repeats ...are maintained by telomerase, which solves the end replication problem. We report that the TTAGGG repeat arrays of mammalian telomeres pose a challenge to the DNA replication machinery, giving rise to replication-dependent defects that resemble those of aphidicolin-induced common fragile sites. Gene deletion experiments showed that efficient duplication of telomeres requires the shelterin component TRF1. Without TRF1, telomeres activate the ATR kinase in S phase and show a fragile-site phenotype in metaphase. Single-molecule analysis of replicating telomeres showed that TRF1 promotes efficient replication of TTAGGG repeats and prevents fork stalling. Two helicases implicated in the removal of G4 DNA structures, BLM and RTEL1, were required to repress the fragile-telomere phenotype. These results identify a second telomere replication problem that is solved by the shelterin component TRF1.
Telomeres protect chromosome from degradation and loss of vital sequence, block end-end fusion, and allow the cell to distinguish between broken ends and chromosome ends. Mammalian telomeres end in ...single-stranded (TTAGGG)-rich 3'-overhangs that are tucked back into the preceding double stranded region to form a T-loop. The end structure of mammalian telomeres has just started to be elucidated and through this extra views we highlight one aspect of that structure. We have recently identified the terminal nucleotides of both the C-rich and G-rich telomere strands in human cells and showed that ~ 80 % of the C-rich strands terminate precisely in ATC-5', while the last base of the G-strand is less precise. This finding has important implications for the processing events that act on the telomere ends post-replication. While the mechanism behind this phenotype is yet to be unraveled, we discuss potential models that could explain the last base specificity.
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