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1.
Cell ; 186(15): 3208-3226.e27, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37379838

RESUMO

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.


Assuntos
DNA Helicases , RNA Helicases , DNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Helicases/metabolismo , Grânulos de Estresse , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação ao GTP/metabolismo , RNA Mensageiro/metabolismo , Grânulos Citoplasmáticos/metabolismo
2.
Cell ; 184(3): 655-674.e27, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497611

RESUMO

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo
3.
Cell ; 181(2): 325-345.e28, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302571

RESUMO

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilação , RNA/metabolismo
4.
Cell ; 181(2): 346-361.e17, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302572

RESUMO

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
5.
Nat Immunol ; 20(1): 18-28, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510222

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a key sensor responsible for cytosolic DNA detection. Here we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for DNA sensing and efficient activation of cGAS. G3BP1 enhanced DNA binding of cGAS by promoting the formation of large cGAS complexes. G3BP1 deficiency led to inefficient DNA binding by cGAS and inhibited cGAS-dependent interferon (IFN) production. The G3BP1 inhibitor epigallocatechin gallate (EGCG) disrupted existing G3BP1-cGAS complexes and inhibited DNA-triggered cGAS activation, thereby blocking DNA-induced IFN production both in vivo and in vitro. EGCG administration blunted self DNA-induced autoinflammatory responses in an Aicardi-Goutières syndrome (AGS) mouse model and reduced IFN-stimulated gene expression in cells from a patient with AGS. Thus, our study reveals that G3BP1 physically interacts with and primes cGAS for efficient activation. Furthermore, EGCG-mediated inhibition of G3BP1 provides a potential treatment for cGAS-related autoimmune diseases.


Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , DNA Helicases/metabolismo , Complexos Multiproteicos/metabolismo , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/genética , Catequina/análogos & derivados , Catequina/uso terapêutico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citosol/imunologia , Citosol/metabolismo , DNA/imunologia , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Modelos Animais de Doenças , Exodesoxirribonucleases/genética , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso/tratamento farmacológico , Malformações do Sistema Nervoso/genética , Fármacos Neuroprotetores/uso terapêutico , Fosfoproteínas/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genética
6.
Mol Cell ; 83(7): 1180-1196.e8, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-37028415

RESUMO

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication.


Assuntos
DNA Helicases , RNA Helicases , Humanos , DNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Grânulos de Estresse , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Imunidade Inata , Inflamação/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Transporte/metabolismo
7.
Mol Cell ; 82(1): 140-158.e12, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34890565

RESUMO

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Neoplasias/enzimologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Complexos Multienzimáticos , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ratos
8.
Mol Cell ; 81(1): 198-211.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33296677

RESUMO

Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , Genoma Humano , Instabilidade Genômica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , DNA Helicases/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Mol Cell ; 81(22): 4692-4708.e9, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34555355

RESUMO

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.


Assuntos
Proteína BRCA1/genética , DNA Ligase Dependente de ATP/genética , DNA de Cadeia Simples , Proteína Homóloga a MRE11/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Sistemas CRISPR-Cas , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Feminino , Humanos , Lentivirus/genética , Neoplasias Mamárias Animais , Camundongos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Interferente Pequeno/metabolismo , Transgenes
10.
Mol Cell ; 81(5): 1027-1042.e4, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33453166

RESUMO

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Retroalimentação Fisiológica , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Inibidoras de STAT Ativados/genética , RNA Helicases/genética , Homeostase do Telômero , Telômero/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/antagonistas & inibidores , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/antagonistas & inibidores , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Transdução de Sinais , Sumoilação , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética
11.
Mol Cell ; 78(1): 70-84.e6, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32017897

RESUMO

Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.


Assuntos
Regiões 3' não Traduzidas , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Pareamento de Bases , Linhagem Celular , Regulação da Expressão Gênica , Humanos , RNA Circular/química , RNA Circular/metabolismo
12.
Mol Cell ; 78(6): 1166-1177.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32497495

RESUMO

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Alelos , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA/fisiologia , Humanos , Mutagênese/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
13.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
14.
Mol Cell ; 80(4): 682-698.e7, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33152268

RESUMO

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Quinase 2 Dependente de Ciclina/metabolismo , DNA Polimerase II/metabolismo , Instabilidade Genômica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ciclo Celular , Proliferação de Células , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA , DNA Polimerase II/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Transdução de Sinais , Células Tumorais Cultivadas
15.
Mol Cell ; 77(6): 1193-1205.e5, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31981475

RESUMO

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.


Assuntos
DNA Helicases/metabolismo , Lisossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA Helicases/genética , Células HEK293 , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , RNA Ribossômico 18S , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Ubiquitina Tiolesterase/genética , Ubiquitinação
16.
Nat Methods ; 21(2): 247-258, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38200227

RESUMO

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.


Assuntos
RNA , Transcrição Reversa , RNA/genética , RNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação/genética , Ligação Proteica
17.
Nature ; 599(7884): 308-314, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34671165

RESUMO

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.


Assuntos
Apoptose , Fragmentação do DNA , DNA Circular/biossíntese , DNA Circular/imunologia , Imunidade Inata , Animais , Células Cultivadas , Mapeamento Cromossômico , DNA Ligase Dependente de ATP/metabolismo , DNA Circular/genética , DNA Circular/isolamento & purificação , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Genoma/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
18.
Nature ; 598(7880): 368-372, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34526721

RESUMO

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.


Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Gênica , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
19.
Proc Natl Acad Sci U S A ; 121(25): e2320995121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38865271

RESUMO

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.


Assuntos
Pareamento Cromossômico , Troca Genética , Meiose , Ubiquitina-Proteína Ligases , Animais , Camundongos , Masculino , Feminino , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Camundongos Knockout , Humanos , Ligases
20.
Proc Natl Acad Sci U S A ; 121(24): e2404383121, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38843184

RESUMO

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.


Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Humanos , Animais , Camundongos , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Transcrição Gênica , Fosforilação , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Camundongos Knockout , Dano ao DNA , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Cromatina/metabolismo , Ubiquitinação , Reparo por Excisão
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