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Neurofibromatosis Type 1 (NF1) is one of the most common genetically inherited disorders that affects 1 in 3000 children annually. Clinical manifestations vary widely but nearly always include the development of cutaneous, plexiform and diffuse neurofibromas that are managed over many years. Recent single-cell transcriptomics profiling efforts of neurofibromas have begun to reveal cell signaling processes. However, the cell signaling networks in mature, non-cutaneous neurofibromas remain unexplored. Here, we present insights into the cellular composition and signaling within mature neurofibromas, contrasting with normal adjacent tissue, in a porcine model of NF1 using single-cell RNA sequencing (scRNA-seq) analysis and histopathological characterization. These neurofibromas exhibited classic diffuse-type histologic morphology and expected patterns of S100, SOX10, GFAP, and CD34 immunohistochemistry. The porcine mature neurofibromas closely resemble human neurofibromas histologically and contain all known cellular components of their human counterparts. The scRNA-seq confirmed the presence of all expected cell types within these neurofibromas and identified novel populations of fibroblasts and immune cells, which may contribute to the tumor microenvironment by suppressing inflammation, promoting M2 macrophage polarization, increasing fibrosis, and driving the proliferation of Schwann cells. Notably, we identified tumor-associated IDO1 +/CD274+ (PD-L1) + dendritic cells, which represent the first such observation in any NF1 animal model and suggest the role of the upregulation of immune checkpoints in mature neurofibromas. Finally, we observed that cell types in the tumor microenvironment are poised to promote immune evasion, extracellular matrix reconstruction, and nerve regeneration.
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Misalignment of the circadian clock compared to environmental cues causes circadian desynchrony, which is pervasive in humans. Clock misalignment can lead to various pathologies including obesity and diabetes, both of which are associated with pancreatic ductal adenocarcinoma - a devastating cancer with an 80% five-year mortality rate. Although circadian desynchrony is associated with an increased risk of several solid-organ cancers, the correlation between clock misalignment and pancreas cancer is unclear. Using a chronic jetlag model, we investigated the impact of clock misalignment on pancreas cancer initiation in mice harboring a pancreas-specific activated Kras mutation. We found that chronic jetlag accelerated the development of pancreatic cancer precursor lesions, with a concomitant increase in precursor lesion grade. Cell-autonomous knock-out of the clock in pancreatic epithelial cells of Kras-mutant mice demonstrated no acceleration of precursor lesion formation, indicating non-cell-autonomous clock dysfunction was responsible for the expedited tumor development. Therefore, we applied single-cell RNA sequencing over time and identified fibroblasts as the cell population manifesting the greatest clock-dependent changes, with enrichment of specific cancer-associated fibroblast pathways due to circadian misalignment.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Ritmo Circadiano/genética , Obesidad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites. Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.
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INTRODUCTION: Prior to the 2018 publication of the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC), a Web-based interobserver study was performed to evaluate MSRSGC reporting categories, identify cytomorphologic features that represent poor sources of agreement, and establish a baseline for future studies. MATERIAL AND METHODS: Study participants evaluated 75 images chosen from the MSRSGC image set, prior to the release of the Milan Atlas. Images spanned all diagnostic categories including typical and borderline cytomorphology. Participant demographics were collected on level of training, practice patterns, and experience. RESULTS: A total of 647 persons attempted access to the survey. Of these, 555 correctly answered the qualifying questions. Participants included: 16.5% ASCP Certified Cytotechnologists, 2.8% Specialist Cytotechnologists, 5.8% IAC Certified individuals, 14.3% Anatomic (AP) Certified Pathologists, 38.9% AP and Cytopathology Certified Pathologists, and 15.3% pathology trainees. Length of participant practice varied from 0 to 54 years. In our sample, 43.4% of participants came from academic centers, 17.6% from private hospitals; and 13.3% from commercial/private laboratories. Overall, 42% of respondents agreed with the reference interpretations of salivary gland lesions. The best agreement was seen in cytopathology certified pathologists. Among the MSRSGC categories, best agreement was found in Neoplasm-Benign (58.9%) and Non-Diagnostic (49.2%) categories, followed by Malignant (48.4%). The agreement rates for Salivary Gland Lesion of Uncertain Malignant Potential (SUMP) and Suspicious For Malignancy (SFM) were 23.6% and 22.7%, respectively. CONCLUSIONS: Similar to the reproducibility studies conducted for gynecologic and urinary cytopathology, the most important factor in diagnostic reproducibility was a priori classification of image difficulty, although people with higher certifications performed better.
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Citodiagnóstico/métodos , Reproducibilidad de los Resultados , Neoplasias de las Glándulas Salivales , Biopsia con Aguja Fina , Humanos , Masculino , Patólogos , Neoplasias de las Glándulas Salivales/diagnóstico , Neoplasias de las Glándulas Salivales/patología , Glándulas Salivales/patologíaRESUMEN
OBJECTIVES: To review rare cases of BK polyomavirus (BKPyV) associated urologic carcinomas in kidney transplant recipients at one institution and in the literature. METHODS: We describe the clinicopathologic features of BKPyV-associated urologic carcinomas in a single-institution cohort. RESULTS: Among 4,772 kidney recipients during 1994 to 2014, 26 (0.5%) and 26 (0.5%) developed posttransplantation urothelial carcinomas (UCs) and renal cell carcinomas (RCCs), respectively, as of 2017. Six (27%) UCs but none of the RCCs expressed large T antigen (TAg). TAg-expressing UCs were high grade with p16 and p53 overexpression (P < .05 compared to TAg-negative UCs). Tumor genome sequencing revealed BKPyV integration and a lack of pathogenic mutations in 50 cancer-relevant genes. Compared to TAg-negative UCs, TAg-expressing UCs more frequently presented at advanced stages (50% T3-T4) with lymph node involvement (50%) and higher UC-specific mortality (50%). CONCLUSIONS: Post-renal transplantation BKPyV-associated UCs are aggressive and genetically distinct from most non-BKPyV-related UCs.
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Carcinoma de Células Renales/patología , Carcinoma de Células Transicionales/patología , Neoplasias Renales/patología , Trasplante de Riñón/efectos adversos , Infecciones por Polyomavirus/patología , Infecciones Tumorales por Virus/patología , Adulto , Anciano , Virus BK , Carcinoma de Células Renales/etiología , Carcinoma de Células Transicionales/etiología , Femenino , Humanos , Riñón/patología , Neoplasias Renales/etiología , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/etiología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/patología , Estudios Retrospectivos , Infecciones Tumorales por Virus/etiologíaRESUMEN
Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC-1a is induced by CR in multiple tissues, and at the cellular level, CR-like activation of PGC-1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC-1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post-transcriptional and post-translational mechanisms were also identified as outcomes of PGC-1a-directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC-1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC-1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities.
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Restricción Calórica , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Células 3T3-L1 , Animales , Células Cultivadas , Senescencia Celular , Metabolismo Energético , Ratones , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genéticaRESUMEN
OBJECTIVE: Charcot-Marie-Tooth (CMT) disease is most commonly caused by duplication of a chromosomal segment surrounding Peripheral Myelin Protein 22, or PMP22 gene, which is classified as CMT1A. Several candidate therapies reduce Pmp22 mRNA levels in CMT1A rodent models, but development of biomarkers for clinical trials in CMT1A is a challenge given its slow progression and difficulty in obtaining nerve samples. Quantitative PCR measurements of PMP22 mRNA in dermal nerves were performed using skin biopsies in human clinical trials for CMT1A, but this approach did not show increased PMP22 mRNA in CMT1A patients compared to controls. One complicating factor is the variable amounts of Schwann cells (SCs) in skin. The objective of the study was to develop a novel method for precise evaluation of PMP22 levels in skin biopsies that can discriminate CMT1A patients from controls. METHODS: We have developed methods to normalize PMP22 transcript levels to SC-specific genes that are not altered by CMT1A status. Several CMT1A-associated genes were assembled into a custom Nanostring panel to enable precise transcript measurements that can be normalized to variable SC content. RESULTS: The digital expression data from Nanostring analysis showed reproducible elevation of PMP22 levels in CMT1A versus control skin biopsies, particularly after normalization to SC-specific genes. INTERPRETATION: This platform should be useful in clinical trials for CMT1A as a biomarker of target engagement that can be used to optimize dosing, and the same normalization framework is applicable to other types of CMT. ANN NEUROL 2019;85:887-898.
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Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Proteínas de la Mielina/genética , Células de Schwann/patología , Piel/patología , Adolescente , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Biopsia , Enfermedad de Charcot-Marie-Tooth/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas de la Mielina/biosíntesis , Células de Schwann/metabolismo , Piel/metabolismo , Adulto JovenRESUMEN
PURPOSE: We aimed to estimate the carrier frequency of Zellweger spectrum disorder (ZSD), a rare autosomal recessive disease, and the associated disease incidence based on data from the Exome Aggregation Consortium (ExAC) of approximately 60,000 individuals. METHODS: We obtained variants from ExAC in 13 PEX genes associated with ZSD. Potentially pathogenic missense variants were identified with computational variant analysis tools according to three stringency levels. Using variants classified as potentially pathogenic, we estimated the carrier frequency and the associated incidence for the entire ExAC population and its subpopulations. We also evaluated variants based on pathogenicity criteria for sequence variant interpretation outlined by the American College of Medical Genetics and Genomics (ACMG) and calculated the carrier frequency and incidence based on those variants. RESULTS: The bioinformatically estimated incidence rate of ZSD in the ExAC population is 1 in 83,841 using our least stringent pathogenicity cutoff. Under clinical guidelines outlined by ACMG, the estimated incidence is 1 in 3,275,751 births. CONCLUSION: We outlined a process for estimating the ZSD disease carrier frequency and incidence in a large consortium using bioinformatics tools. Our results are close to current newborn screening estimates in New York of 1 in 90,000 births, estimated from 1.08 million screenings.
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Exoma/genética , Tamización de Portadores Genéticos/métodos , Predisposición Genética a la Enfermedad , Síndrome de Zellweger/diagnóstico , Biología Computacional , Bases de Datos Genéticas , Variación Genética , Humanos , Recién Nacido , Mutación , Tamizaje Neonatal/métodos , Síndrome de Zellweger/epidemiología , Síndrome de Zellweger/genéticaRESUMEN
Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.
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Caloric restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here, we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic metabolism. We conducted molecular profiling of liver biopsies collected from adult male rhesus monkeys (Macaca mulatta) at baseline and after 2 years on control or CR (30% restricted) diet. Quantitation of over 20,000 molecules from the hepatic transcriptome, proteome, and metabolome indicated that metabolism and RNA processing are major features of the response to CR. Predictive models identified lipid, branched-chain amino acid, and short-chain carbon metabolic pathways, with alternate transcript use for over half of the genes in the CR network. We conclude that RNA-based mechanisms are central to the CR response and integral in metabolic reprogramming.
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Restricción Calórica , Hígado/metabolismo , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Envejecimiento/metabolismo , Animales , Expresión Génica , Macaca mulatta , MasculinoRESUMEN
OBJECTIVES: In concert with the 2015 publication of The Paris System for Urinary Cytopathology (TPS), a Web-based interobserver study, co-sponsored by the American Society of Cytopathology (ASC) and International Academy of Cytology (IAC), was performed to determine diagnostic agreement among volunteer participants and with the TPS author consensus. MATERIAL AND METHODS: Participants at various levels of training and certification were recruited through national and international cytopathology professional societies. Although the survey was open to all comers, potential participants were screened by two basic cytopathology questions. Information was collected on the level of training, practice patterns, and experience. Study participants evaluated 85 images (previously unpublished) chosen from the TPS atlas. These images spanned all diagnostic categories. RESULTS: Of the 1993 attempts to access the survey, 1313 participants correctly answered the qualifying questions and were included in the survey. Respondents were concentrated in the United States, although many participants came from other countries. The majority of respondents were board-certified in anatomic pathology with cytopathology certification. A smaller number were cytotechnologists. Board-certified cytopathologists and specialist cytotechnologists outperformed other certifications. Practice type (academics versus non-academic), and country (US versus international) were not major factors in concordance. Diagnostic categories with the best agreement were Negative for High-Grade Urothelial Carcinoma (NHGUC; 71%), Low-Grade Urothelial Neoplasm (LGUN; 62%), and High-Grade Urothelial Carcinoma (HGUC; 57%). Indeterminate categories showed low concordance. CONCLUSIONS: The NHGUC, LGUN, and HGUC were most correlated with diagnostic agreement among observers. This study can serve as a baseline for future comparisons.
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Purpose: To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. Methods: Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sanger sequencing was used to confirm variants in original DNA, and to test for disease cosegregation in additional family members. Candidate genes and chromosomal loci previously associated with myopic refractive error and its endophenotypes were comprehensively screened. Results: In 14 high myopia families, we identified 73 rare and 31 novel gene variants as candidates for pathogenicity. In seven of these families, two of the novel and eight of the rare variants were within known myopia loci. A total of 104 heterozygous nonsynonymous rare variants in 104 genes were identified in 10 out of 14 probands. Each variant cosegregated with affection status. No rare variants were identified in genes known to cause myopia or in genes closest to published genome-wide association study association signals for refractive error or its endophenotypes. Conclusions: Whole exome sequencing was performed to determine gene variants implicated in the pathogenesis of AD high myopia. This study provides new genes for consideration in the pathogenesis of high myopia, and may aid in the development of genetic profiling of those at greatest risk for attendant ocular morbidities of this disorder.
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ADN/genética , Exoma/genética , Proteínas del Ojo/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/métodos , Mutación , Miopía/genética , Análisis Mutacional de ADN , Proteínas del Ojo/metabolismo , Femenino , Humanos , Masculino , LinajeRESUMEN
INTRODUCTION: In concert with the 2014 update to the Bethesda System for Reporting Cervical Cytology, a Web-based image interobserver study was performed to evaluate concordance with the "expert panel" interpretation, as was done during the Bethesda 2001 update. The aim was to identify cytomorphologic features and Bethesda reporting categories that represent sources of poor interobserver agreement and see how the trends compared to the first Bethesda Interobserver Reproducibility Study (BIRST). MATERIALS AND METHODS: Participants were recruited online through national and international cytopathology professional societies. Study participants evaluated 84 previously unpublished web images chosen from the third Bethesda Atlas image set, prior to the release of the atlas. These images spanned all reporting categories and included typical and borderline cytomorphology. Demographic information was collected on level of training, practice patterns, and experience of the participants. Participation was restricted to those correctly answering 2 basic cytopathology questions, ensuring minimal knowledge of gynecologic cytopathology. RESULTS: A total of 1290 unique individuals attempted access to this Web-based study and 833 correctly answered the two qualifying questions. Of these, 518 respondents completed the survey. Participant origin included: 59% United States, 41% international; 48% cytotechnologists, 41% pathologists, 5% fellows, and 6% other. Practice types were: 39% academic institutions, 29% private hospitals, and 16% commercial laboratories. Overall, the mean participant agreement with the exact Bethesda panel interpretation was 62.8%. The best agreement was found for negative for intraepithelial lesion or malignancy (NILM; 74%) and low-grade squamous intraepithelial lesion (LSIL; 86%) categories. Squamous cell carcinoma (SCC) (63%), high-grade squamous intraepithelial lesion (HSIL; 60%), atypical squamous cells of undetermined significance (ASC-US; 62%) and atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H; 60%) showed slightly lower concordance with the panel interpretations. Cervical glandular lesions were more problematic (33%). Anal samples performed similarly to their gynecologic counterparts. There was similar diagnostic agreement across participant certifications and practice type (academic versus non-academic). Performance was higher for United States and other North America-based participants (P = 0.0104). This significance may be attributed to a language bias, as the survey was only offered in English. CONCLUSIONS: Similar to the BIRST-1 study conducted in 2001, the most important factor for diagnostic agreement by cytotechnologists, pathologists, and trainees was the a priori difficulty of an image rather than participant training, certification, or experience. Participants showed better general diagnostic agreement with the expert panel interpretations of the material in BIRST-2 than in BIRST-1. Agreement was highest for Bethesda categories of NILM, LSIL, HSIL, and SCC. Concordance for even the borderline ASC-US and ASC-H categories exhibited remarkable improvement in the BIRST-2.
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Congenital insensitivity to pain with anhidrosis is a rare autosomal recessive disorder. It has been reported that the defect in the NTRK1 gene encoding tropomyosin-related kinase A (TrkA) can cause congenital insensitivity to pain with anhidrosis. Nerve growth factor (NGF), the product of NGFB, mediates biological effects by binding to and activating tropomyosin-related kinase A. In addition, necdin (encoded by NDN) is also essential in nerve growth factor-tropomyosin-related kinase A pathway. We performed mutation analysis in NTRK1, NGFB, and NDN genes in a Chinese Han 17-year-old female patient with congenital insensitivity to pain with anhidrosis and her healthy family members. As a result, the patient was found to have a novel insertion in exon 7 (c.727insT) of NTRK1, which causes premature termination, and a single nucleotide polymorphism (rs2192206 G>A) in NDN. Our findings imply that the genetic variations of the nerve growth factor-tropomyosin-related kinase A pathway play an important role in congenital insensitivity to pain with anhidrosis.
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Mutación del Sistema de Lectura , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Receptor trkA/genética , Adolescente , Pueblo Asiatico/genética , China , Análisis Mutacional de ADN , Femenino , Neuropatías Hereditarias Sensoriales y Autónomas/patología , Neuropatías Hereditarias Sensoriales y Autónomas/fisiopatología , Humanos , LinajeRESUMEN
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei. Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3' to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3' to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.
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Caenorhabditis elegans/genética , Interferencia de ARN , ARN Polimerasa II/antagonistas & inhibidores , ARN de Helminto/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Secuencia Conservada , Genes de Helminto/genética , Unión Proteica , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN de Helminto/biosíntesis , ARN de Helminto/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/genéticaRESUMEN
Years after the discovery that Dicer is a key enzyme in gene silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in Caenorhabditis elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not necessary for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wild-type and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26-nucleotide small RNA species containing a 5' guanosine.
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Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Eliminación de Gen , Genes de Helminto , MicroARNs/genética , MicroARNs/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasa III/genética , Homología de Secuencia de AminoácidoRESUMEN
Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated ("ERI") factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Factores Eucarióticos de Iniciación/metabolismo , Exorribonucleasas/metabolismo , Mutación , Fosforilación , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , Ribonucleasa III/metabolismoRESUMEN
Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis/citología , Caenorhabditis/genética , Exorribonucleasas/metabolismo , Interferencia de ARN/fisiología , Ribonucleasa III/metabolismo , Espermatogénesis , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis/metabolismo , Proteínas de Caenorhabditis elegans/química , Exorribonucleasas/química , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Ribonucleasa III/química , Espermatozoides/citologíaRESUMEN
Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.