RESUMO
Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.
Assuntos
Nucléolo Celular/enzimologia , Nucléolo Celular/genética , DNA Ribossômico/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Ribossomos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/fisiologia , DNA Helicases/metabolismo , DNA Intergênico/genética , Humanos , Enzimas Multifuncionais/metabolismo , Biossíntese de Proteínas , Estruturas R-Loop , RNA Helicases/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Ribonuclease H/metabolismo , Ribossomos/química , Ribossomos/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.
Assuntos
Proteína BRCA1 , Replicação do DNA , Síndrome Hereditária de Câncer de Mama e Ovário , Mutação , Transcrição Gênica , Humanos , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Replicação do DNA/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Síndrome Hereditária de Câncer de Mama e Ovário/patologia , Síndrome Hereditária de Câncer de Mama e Ovário/fisiopatologia , RNA Polimerase II/metabolismo , Transcrição Gênica/genética , Regiões Promotoras Genéticas , Metiltransferases/deficiência , Metiltransferases/genética , Estruturas R-Loop , Morte CelularRESUMO
Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.
Assuntos
Proteína BRCA1 , Neoplasias da Mama , Animais , Feminino , Humanos , Camundongos , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Neoplasias da Mama/genética , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Instabilidade Genômica , Recidiva Local de Neoplasia , Estruturas R-Loop , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/metabolismo , Locomoção/fisiologia , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Mutação/genética , Neurônios , Fosfoproteínas/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Purpose This study examines the outcomes of locally advanced head and neck squamous cell carcinoma (HNSCC) following the adoption of conventional intensity-modulated radiotherapy (cIMRT) and volumetric-modulated arc therapy (VMAT) over a decade. The region under study has higher comorbidities associated with increased HNSCC incidence and poorer prognosis. Materials and methods A 10-year retrospective review of electronic medical records included 296 patients with stage III, IVA, and IVB HNSCC (American Joint Committee on Cancer, Seventh edition). Survival outcomes were compared between VMAT and cIMRT using Kaplan-Meier survival curves and adjusted for relevant demographic factors using Cox's proportional hazards model. Analysis was performed using R software (R Foundation, Vienna, Austria). Results The median age of the cohort was 63 years, comprising of 80% males. The oropharynx was the most common primary tumor site. 264 (89%) received 50Gy or higher dose radiation by either cIMRT (22%) or VMAT (67%). At five years, locoregional control (LC) and overall survival (OS) rates were 79.5% and 56.7%, respectively. VMAT showed a significant improvement in five-year OS (63.4% versus 43.8% for cIMRT, p=0.0023) but no significant difference in five-year LC (81% VMAT versus 74.5% cIMRT, p=0.17). Grade 3-4 acute toxicity was observed in 22% of patients. Conclusions VMAT and cIMRT demonstrated excellent LC in locally advanced HNSCC despite high comorbidity rates. Notably, VMAT was associated with significantly better OS compared to cIMRT. These outcomes surpass historical data, suggesting that VMAT technology may lead to improved patient outcomes. However, larger randomized controlled trials and dosimetric studies are needed to confirm these findings.
RESUMO
Pathogenic variants in the BRCA1 and BRCA2 genes are associated with increased risk for breast and ovarian cancers. Concurrent mutations in both genes in the same individual are rare but pose specific challenges when identified, usually through multigene panel testing or infrequently from a genome-wide analysis, such as whole-exome sequencing (WES). We present a 15-year-old female patient with syndromic intellectual disability whose exome reanalysis identified secondary findings of pathogenic BRCA1 and BRCA2 variants, both inherited paternally. We discuss the significant challenges posed by this finding in genetic counseling and cancer risk management of an adolescent with nonverbal intellectual disability, as well as the impact on their family. This rare case highlights the potential increased diagnostic yield of whole exome sequencing reanalysis and the consequences of secondary medically actionable results in a pediatric patient.
RESUMO
AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin ß family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.
Assuntos
Leucemia Mieloide Aguda , beta Carioferinas , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
The principle of synthetic lethality, which refers to the loss of viability resulting from the disruption of two genes, which, individually, do not cause lethality, has become an attractive target approach due to the development and clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this review, we present the most recent findings on the use of PARPi in the clinic, which are currently approved for second-line therapy for advanced ovarian and breast cancer associated with mutations of BRCA1 or BRCA2 (BRCA1/2) genes. PARPi efficacy, however, appears to be limited by acquired and inherent resistance, highlighting the need for alternative and synergistic targets to eliminate these tumors. Here, we explore other identified synthetic lethal interactors of BRCA1/2, including DNA polymerase theta (POLQ), Fanconi anemia complementation group D2 (FANDC2), radiation sensitive 52 (RAD52), Flap structure-specific endonuclease 1 (FEN1), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APE2), as well as other protein and nonprotein targets, for BRCA1/2-mutated cancers and their implications for future therapies. A wealth of information now exists for phenotypic and functional characterization of these novel synthetic lethal interactors of BRCA1/2, and leveraging these findings can pave the way for the development of new targeted therapies for patients suffering from these cancers.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Mutações Sintéticas Letais , HumanosRESUMO
Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.