RESUMO
MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.
Assuntos
Interferon gama/metabolismo , MicroRNAs/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/imunologia , Proteínas/genética , Proteínas de Ligação a RNA , Proteínas com Domínio T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma , Edição de RNA/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA/genética , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Fatores de Transcrição/genéticaRESUMO
MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.
Assuntos
Genes/genética , Genoma/genética , MicroRNAs/genética , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Sequências Repetidas Invertidas/genética , Camundongos , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Ribonuclease III/metabolismoRESUMO
Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to â¼1%-8%. Interestingly, ZFNickases targeting the AAVS1 "safe harbor" locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition-suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Marcação de Genes/métodos , Genoma Humano , Proteínas Recombinantes de Fusão/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular Transformada , Linhagem Celular Tumoral , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA por Junção de Extremidades , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos , Histonas/metabolismo , Humanos , Mutação INDEL , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
OBJECTIVE: The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test for 5 microdeletion syndromes. STUDY DESIGN: Four hundred sixty-nine samples (358 plasma samples from pregnant women, 111 artificial plasma mixtures) were amplified with the use of a massively multiplexed polymerase chain reaction, sequenced, and analyzed with the use of the Next-generation Aneuploidy Test Using SNPs algorithm for the presence or absence of deletions of 22q11.2, 1p36, distal 5p, and the Prader-Willi/Angelman region. RESULTS: Detection rates were 97.8% for a 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1), and cri-du-chat syndromes (24/24). False-positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442), or 1p36 deletion syndromes (0/422). CONCLUSION: SNP-based noninvasive prenatal microdeletion screening is highly accurate. Because clinically relevant microdeletions and duplications occur in >1% of pregnancies, regardless of maternal age, noninvasive screening for the general pregnant population should be considered.
Assuntos
Deleção Cromossômica , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Testes para Triagem do Soro Materno , Polimorfismo de Nucleotídeo Único , Algoritmos , Transtornos Cromossômicos/genética , Reações Falso-Positivas , Feminino , Humanos , Reação em Cadeia da Polimerase Multiplex , Valor Preditivo dos Testes , Gravidez , Reprodutibilidade dos Testes , Análise de Sequência de DNA , SíndromeRESUMO
Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity.
Assuntos
Infecções por Citomegalovirus/imunologia , Exonucleases/genética , Exonucleases/imunologia , Células Matadoras Naturais/imunologia , MicroRNAs/imunologia , Transferência Adotiva , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Exorribonucleases , Homeostase/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Mutantes , MicroRNAs/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismoRESUMO
Noncanonical microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) are distinct subclasses of small RNAs that bypass the DGCR8/DROSHA Microprocessor but still require DICER1 for their biogenesis. What role, if any, they have in mammals remains unknown. To identify potential functional properties for these subclasses, we compared the phenotypes resulting from conditional deletion of Dgcr8 versus Dicer1 in post-mitotic neurons. The loss of Dicer1 resulted in an earlier lethality, more severe structural abnormalities, and increased apoptosis relative to that from Dgcr8 loss. Deep sequencing of small RNAs from the hippocampus and cortex of the conditional knockouts and control littermates identified multiple noncanonical microRNAs that were expressed at high levels in the brain relative to other tissues, including mirtrons and H/ACA snoRNA-derived small RNAs. In contrast, we found no evidence for endo-siRNAs in the brain. Taken together, our findings provide evidence for a diverse population of highly expressed noncanonical miRNAs that together are likely to play important functional roles in post-mitotic neurons.
Assuntos
Encéfalo/metabolismo , RNA Helicases DEAD-box/metabolismo , MicroRNAs/genética , Proteínas/metabolismo , Ribonuclease III/metabolismo , Animais , Encéfalo/citologia , RNA Helicases DEAD-box/deficiência , Camundongos , Camundongos Knockout , Mitose , Fenótipo , Proteínas de Ligação a RNA , Ribonuclease III/deficiência , Análise de Sequência de RNARESUMO
CONTEXT: Broad genomic analyses among thyroid histologies have been described from relatively small cohorts. OBJECTIVE: Investigate the molecular findings across a large, real-world cohort of thyroid fine-needle aspiration (FNA) samples. DESIGN: Retrospective analysis of RNA sequencing data files. SETTING: Clinical Laboratory Improvement Amendments laboratory performing Afirma Genomic Sequencing Classifier (GSC) and Xpression Atlas (XA) testing. PARTICIPANTS: A total of 50 644 consecutive Bethesda III-VI nodules. INTERVENTION: None. MAIN OUTCOME MEASURES: Molecular test results. RESULTS: Of 48 952 Bethesda III/IV FNAs studied, 66% were benign by Afirma GSC. The prevalence of BRAF V600E was 2% among all Bethesda III/IV FNAs and 76% among Bethesda VI FNAs. Fusions involving NTRK, RET, BRAF, and ALK were most prevalent in Bethesda V (10%), and 130 different gene partners were identified. Among small consecutive Bethesda III/IV sample cohorts with one of these fusions and available surgical pathology excision data, the positive predictive value of an NTRK or RET fusion for carcinoma or noninvasive follicular thyroid neoplasm with papillary-like nuclear features was >95%, whereas for BRAF and ALK fusions it was 81% and 67%, respectively. At least 1 genomic alteration was identified by the expanded Afirma XA panel in 70% of medullary thyroid carcinoma classifier-positive FNAs, 44% of Bethesda III or IV Afirma GSC suspicious FNAs, 64% of Bethesda V FNAs, and 87% of Bethesda VI FNAs. CONCLUSIONS: This large study demonstrates that almost one-half of Bethesda III/IV Afirma GSC suspicious and most Bethesda V/VI nodules had at least 1 genomic variant or fusion identified, which may optimize personalized treatment decisions.
Assuntos
Proteínas Proto-Oncogênicas B-raf/genética , Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/genética , Quinase do Linfoma Anaplásico/genética , Feminino , Perfilação da Expressão Gênica , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-ret/genética , Receptor trkA/genética , Estudos Retrospectivos , Nódulo da Glândula Tireoide/patologiaRESUMO
Heterochromatin renders domains of chromosomes transcriptionally silent and, due to clonal variation in its formation, can generate heritably distinct populations of genetically identical cells. Saccharomyces cerevisiae's Sir1 functions primarily in the establishment, but not the maintenance, of heterochromatic silencing at the HMR and HML loci. In several Saccharomyces species, we discovered multiple paralogs of Sir1, called Kos1-Kos4 (Kin of Sir1). The Kos and Sir1 proteins contributed partially overlapping functions to silencing of both cryptic mating loci in S. bayanus. Mutants of these paralogs reduced silencing at HML more than at HMR. Most genes of the SIR1 family were located near telomeres, and at least one paralog was regulated by telomere position effect. In S. cerevisiae, Sir1 is recruited to the silencers at HML and HMR via its ORC interacting region (OIR), which binds the bromo adjacent homology (BAH) domain of Orc1. Zygosaccharomyces rouxii, which diverged from Saccharomyces after the appearance of the silent mating cassettes, but before the whole-genome duplication, contained an ortholog of Kos3 that was apparently the archetypal member of the family, with only one OIR. In contrast, a duplication of this domain was present in all orthologs of Sir1, Kos1, Kos2, and Kos4. We propose that the functional specialization of Sir3, itself a paralog of Orc1, as a silencing protein was facilitated by the tandem duplication of the OIR domain in the Sir1 family, allowing distinct Sir1-Sir3 and Sir1-Orc1 interactions through OIR-BAH domain interactions.
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Variação Genética/fisiologia , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Teste de Complementação Genética , Modelos Biológicos , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/fisiologia , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Background: Fine needle aspiration (FNA) cytology, a diagnostic test central to thyroid nodule management, may yield indeterminate results in up to 30% of cases. The Afirma® Genomic Sequencing Classifier (GSC) was developed and clinically validated to utilize genomic material obtained during the FNA to accurately identify benign nodules among those deemed cytologically indeterminate so that diagnostic surgery can be avoided. A key question for diagnostic tests is their robustness under different perturbations that may occur in the lab. Herein, we describe the analytical performance of the Afirma GSC. Results: We examined the analytical sensitivity of the Afirma GSC to varied input RNA amounts and the limit of detection of malignant signals with heterogenous samples mixed with adjacent normal or benign tissues. We also evaluated the analytical specificity from potential interfering substances such as blood and genomic DNA. Further, the inter-laboratory, intra-run, and inter-run reproducibility of the assay were examined. Analytical sensitivity analysis showed that Afirma GSC calls are tolerant to variation in RNA input amount (5-30 ng), and up to 75% dilution of malignant FNA material. Analytical specificity studies demonstrated Afirma GSC remains accurate in presence of up to 75% blood or 30% genomic DNA. The Afirma GSC results are highly reproducible across different operators, runs, reagent lots, and laboratories. Conclusion: The analytical robustness and reproducibility of the Afirma GSC test support its routine clinical use among thyroid nodules with indeterminant FNA cytology.
RESUMO
Introduction: The Afirma® Xpression Atlas (XA) detects gene variants and fusions in thyroid nodule FNA samples from a curated panel of 511 genes using whole-transcriptome RNA-sequencing. Its intended use is among cytologically indeterminate nodules that are Afirma GSC suspicious, Bethesda V/VI nodules, or known thyroid metastases. Here we report its analytical and clinical validation. Methods: DNA and RNA were purified from the same sample across 943 blinded FNAs and compared by multiple methodologies, including whole-transcriptome RNA-seq, targeted RNA-seq, and targeted DNA-seq. An additional 695 blinded FNAs were used to define performance for fusions between whole-transcriptome RNA-seq and targeted RNA-seq. We quantified the reproducibility of the whole-transcriptome RNA-seq assay across laboratories and reagent lots. Finally, variants and fusions were compared to histopathology results. Results: Of variants detected in DNA at 5 or 20% variant allele frequency, 74 and 88% were also detected by XA, respectively. XA variant detection was 89% when compared to an alternative RNA-based detection method. Low levels of expression of the DNA allele carrying the variant, compared with the wild-type allele, was found in some variants not detected by XA. 82% of gene fusions detected in a targeted RNA fusion assay were detected by XA. Conversely, nearly all variants or fusions detected by XA were confirmed by an alternative method. Analytical validation studies demonstrated high intra-plate reproducibility (89%-94%), inter-plate reproducibility (86-91%), and inter-lab accuracy (90%). Multiple variants and fusions previously described across the spectrum of thyroid cancers were identified by XA, including some with approved or investigational targeted therapies. Among 190 Bethesda III/IV nodules, the sensitivity of XA as a standalone test was 49%. Conclusion: When the Afirma Genomic Sequencing Classifier (GSC) is used first among Bethesda III/IV nodules as a rule-out test, XA supplements genomic insight among those that are GSC suspicious. Our data clinically and analytically validate XA for use among GSC suspicious, or Bethesda V/VI nodules. Genomic information provided by XA may inform clinical decision-making with precision medicine insights across a broad range of FNA sample types encountered in the care of patients with thyroid nodules and thyroid cancer.
RESUMO
We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.
RESUMO
To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Miócitos Cardíacos/citologia , RNA Mensageiro/metabolismo , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Análise por Conglomerados , Biologia Computacional/métodos , Feto/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Coração/crescimento & desenvolvimento , Humanos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , Fatores de Tempo , Transcrição Gênica , TranscriptomaRESUMO
Improved in vitro systems for predicting drug-induced toxicity are needed in the pharmaceutical and biotechnology industries to decrease late-stage drug attrition. One unmet need is an early screen for cardiotoxicity, which accounts for about one third of safety-based withdrawn pharmaceuticals. Herein, the first published report of a high-throughput functional assay employing a monolayer of beating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is described, detailing a model that accurately detects drug-induced cardiac abnormalities. Using 96-well plates with interdigitated electrode arrays that assess impedance, the rhythmic, synchronous contractions of the iPSC-CMs were detected. Treatment of the iPSC-CMs with 28 different compounds with known cardiac effects resulted in compound-specific changes in the beat rate and/or the amplitude of the impedance measurement. Changes in impedance for the compounds tested were comparable with the results from a related technology, electric field potential assessment obtained from microelectrode arrays. Using the results from the set of compounds, an index of drug-induced arrhythmias was calculated, enabling the determination of a drug's proarrhythmic potential. This system of interrogating human cardiac function in vitro opens new opportunities for predicting cardiac toxicity and studying cardiac biology.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos/efeitos adversos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas/métodos , Humanos , Preparações FarmacêuticasRESUMO
This report demonstrates that introduction of microRNAs (miRNAs) specific to embryonic stem cells enhances the production of mouse induced pluripotent stem (iPS) cells. The miRNAs miR-291-3p, miR-294 and miR-295 increase the efficiency of reprogramming by Oct4, Sox2 and Klf4, but not by these factors plus cMyc. cMyc binds the promoter of the miRNAs, suggesting that they are downstream effectors of cMyc during reprogramming. However, unlike cMyc, the miRNAs induce a homogeneous population of iPS cell colonies.
Assuntos
Desdiferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , MicroRNAs/metabolismo , Animais , Fator 4 Semelhante a Kruppel , Camundongos , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.
Assuntos
RNA Helicases DEAD-box/fisiologia , Células-Tronco Embrionárias/metabolismo , Endorribonucleases/fisiologia , MicroRNAs/metabolismo , Proteínas/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Biologia Computacional , Perfilação da Expressão Gênica , Sequências Repetidas Invertidas , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Ribonuclease III , Homologia de Sequência do Ácido Nucleico , Sequências de Repetição em TandemRESUMO
Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.
Assuntos
Ciclo Celular , Proliferação de Células , MicroRNAs/genética , Animais , Células-Tronco Embrionárias , Camundongos , Proteínas/genética , Proteínas de Ligação a RNARESUMO
SWR1-Com, which is responsible for depositing H2A.Z into chromatin, shares four subunits with the NuA4 histone acetyltransferase complex. This overlap in composition led us to test whether H2A.Z was a substrate of NuA4 in vitro and in vivo. The N-terminal tail of H2A.Z was acetylated in vivo at multiple sites by a combination of NuA4 and SAGA. H2A.Z acetylation was also dependent on SWR1-Com, causing H2A.Z to be efficiently acetylated only when incorporated in chromatin. Unacetylatable H2A.Z mutants were, like wild-type H2A.Z, enriched at heterochromatin boundaries, but were unable to block spreading of heterochromatin. A mutant version of H2A.Z that could not be acetylated, in combination with a mutation in a nonessential gene in the NuA4 complex, caused a pronounced decrease in growth rate. This H2A.Z mutation was lethal in combination with a mutant version of histone H4 that could not be acetylated by NuA4. Taken together, these results show a role for H2A.Z acetylation in restricting silent chromatin, and reveal that acetylation of H2A.Z and H4 can contribute to a common function essential to life.