RESUMO
The study of transcription factors that determine specialised neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electro-physiologically excitable cells that link the oxygen content of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by the Type I cells of the carotid body and recent work has revealed one isoform of the transcription factor HIF, HIF-2α, to have a non-redundant role in the development and function of that organ. Here we show that the activation of HIF-2α, including isolated overexpression alone, is sufficient to induce oxygen chemosensitivity in the otherwise unresponsive adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues that resemble the foetal organ of Zuckerkandl and also manifest oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of classes of genes that are ordinarily characteristic of the carotid body, including G-protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking two major oxygen sensing systems.
RESUMO
Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here, we describe a workflow for measurement of ribosome load of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells reveals extensive translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation are limited, and we do not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage are associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR reveal that specific classes of HIF1A and HIF2A transcriptional target gene manifest different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Renais , Serina-Treonina Quinases TOR , Humanos , Neoplasias Renais/genética , Oxigênio , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.
Assuntos
Proteínas Intrinsicamente Desordenadas , Histona Desmetilases com o Domínio Jumonji , Proteínas de Ciclo Celular/metabolismo , Humanos , Hidroxilação , Proteínas Intrinsicamente Desordenadas/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Domínios Proteicos , Fatores de Transcrição/metabolismoRESUMO
Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Glicólise/genética , Humanos , Camundongos , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Neoplasias PancreáticasRESUMO
Myocardial perfusion and perfusion reserve are diminished in patients with atrial fibrillation (AF). Phase-contrast (PC) cine magnetic resonance imaging (MRI) of the coronary sinus serves as a non-invasive means of quantifying coronary flow reserve (CFR) without any radioactive tracer. The present study aimed to evaluate the utility of PC cine MRI of the coronary sinus for assessing decreased CFR in patients with AF. We studied 362 patients with known or suspected coronary artery disease (CAD) [age 72 ± 9 years; 267 (74%) male; 90 (25%) had AF] and 20 age- and gender-matched control subjects [age 72 ± 9 years, 14 (70%) male]. Using a 1.5-T MR scanner and cardiac coils, blood flow of the coronary sinus (CBF) was quantified by PC cine MRI. CFR was calculated as CBF during adenosine triphosphate infusion divided by CBF at rest. CFR was significantly lower in patients with AF than in those without AF among all patients (n = 362) (2.45 ± 0.42 vs. 2.71 ± 0.58, p < 0.001), in patients with known CAD (n = 155) (2.40 ± 0.46 vs. 2.72 ± 0.58, p = 0.002), and in those with suspected CAD (n = 207) (2.49 ± 0.40 vs. 2.72 ± 0.59, p = 0.007). Significant differences in CFR were found between controls and patients without AF (3.12 ± 0.52 vs. 2.71 ± 0.58, p < 0.001). AF was independently associated with CFR in both known CAD patients [ß = - 0.248, 95% confidence interval (CI): - 0.561 to - 0.119, p = 0.003) and suspected CAD patients (ß = - 0.154, 95% CI - 0.353 to - 0.034, p = 0.018). The presence of AF was related to impaired CFR in both known and suspected CAD patients. PC cine MRI of the coronary sinus can be useful for detecting impaired CFR in patients with AF.
Assuntos
Fibrilação Atrial/diagnóstico , Velocidade do Fluxo Sanguíneo/fisiologia , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Imagem Cinética por Ressonância Magnética/métodos , Idoso , Fibrilação Atrial/fisiopatologia , Feminino , Seguimentos , Humanos , Masculino , Estudos RetrospectivosRESUMO
Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 (Calm3L ) mRNA Calm3L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3L mRNA to distal dendrites.
Assuntos
Regiões 3' não Traduzidas , Calmodulina/genética , Dendritos/metabolismo , Íntrons , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Células HeLa , Hipocampo/citologia , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , RatosRESUMO
The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes â¼7 d and the basic bioinformatic pipeline takes 1 d.
Assuntos
Imunoprecipitação/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Linhagem Celular , Colódio/química , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Membranas Artificiais , Ligação Proteica , RNA/química , Especificidade por SubstratoRESUMO
The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND: The prevalence of severe sclerotic aortic stenosis (ScAS) in those at least â§80 years old has been increasing in Japan; however, the prognosis of these Japanese patients without surgical treatment has not been reported. METHODS AND RESULTS: Ninety consecutive patients with medically treated severe ScAS were prospectively studied. To assess further event-free survival rate (EFSR) from either cardiac (heart failure or cardiac death) events or noncardiac deaths, they were divided into three groups based on aortic valve area (AVA) at the initial diagnosis (group A: AVAâ¦0.6cm(2), group B: 0.6cm(2)Assuntos
Estenose da Valva Aórtica/terapia
, Idoso de 80 Anos ou mais
, Valva Aórtica
, Estenose da Valva Aórtica/mortalidade
, Estenose da Valva Aórtica/patologia
, Causas de Morte
, Feminino
, Insuficiência Cardíaca/mortalidade
, Próteses Valvulares Cardíacas
, Humanos
, Japão
, Masculino
, Análise Multivariada
, Prognóstico
, Estudos Prospectivos
, Fatores de Risco
, Volume Sistólico
RESUMO
Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.
Assuntos
Mitocôndrias/metabolismo , RNA de Transferência de Valina/química , Subunidades Ribossômicas/química , Subunidades Ribossômicas/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/ultraestrutura , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA de Transferência de Valina/análise , Subunidades Ribossômicas/genéticaRESUMO
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
Assuntos
Biblioteca Gênica , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/química , RNA/química , Sítios de Ligação , DNA Circular/química , DNA Circular/genética , Regulação da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , RNA/genética , Proteínas de Ligação a RNA/genética , Raios UltravioletaRESUMO
Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.
Assuntos
Citosina/metabolismo , Metilação de DNA , Metiltransferases/metabolismo , RNA não Traduzido/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Metiltransferases/genética , Dados de Sequência Molecular , RNA/genética , RNA não Traduzido/genética , Transcriptoma , TransfecçãoRESUMO
Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Regulação da Expressão Gênica , Ordem dos Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Ligação Proteica , Isoformas de RNA , Proteína FUS de Ligação a RNA/genéticaRESUMO
UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are methods to study protein-RNA interactions in untreated cells and tissues. Here, we analyzed six published and two novel data sets to confirm that both methods identify protein-RNA cross-link sites, and to identify a slight uridine preference of UV-C-induced cross-linking. Comparing Nova CLIP and iCLIP data revealed that cDNA deletions have a preference for TTT motifs, whereas iCLIP cDNA truncations are more likely to identify clusters of YCAY motifs as the primary Nova binding sites. In conclusion, we demonstrate how each method impacts the analysis of protein-RNA binding specificity.
Assuntos
Antígenos de Neoplasias/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Proteínas do Tecido Nervoso/química , Proteínas de Ligação a RNA/química , RNA/química , Animais , Células COS , Chlorocebus aethiops , Biblioteca Gênica , Células HeLa , Humanos , Metiltransferases/química , Camundongos , Anotação de Sequência Molecular , Antígeno Neuro-Oncológico Ventral , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
A simple method for attaching immunoglobulin G (IgG) on the cell surface was successfully developed for enhancing phagocytosis of apoptotic tumor cells (ATCs) by dendritic cells (DCs) ex vivo. By conjugating with a poly(ethylene glycol) (PEG)-lipid, named the biocompatible anchor for the membrane (BAM), arbitrary IgG could be incorporated into the cell membrane. In particular, when IgG-BAM conjugates were prepared at the optimal molar ratio of IgG to BAM (1 to 20), almost all cells were efficiently modified with IgG by treatment with IgG-BAM. This simple method was successfully applied to four types of mammalian cells. Furthermore, treatment of ATCs with the IgG-BAM conjugate increased the phagocytosis ratio of ATCs by DCs two-fold when compared to no treatment. This phagocytosis-enhancing effect was nearly identical to treatment with a tumor-specific IgG. Thus, without employing the tumor-specific IgG, which is difficult to obtain for any tumor cells and is expensive, the present method could opsonize ATC with the use of arbitrary IgG. The results strongly indicate that IgG-BAM treatment represents a promising method for opsonizing ATC with human serum IgG, and that this approach will lead to objective clinical responses in DC vaccines.