RESUMO
Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.
Assuntos
Metilação de DNA , Escherichia coli , Animais , Camundongos , Escherichia coli/genética , Genoma/genética , DNA/metabolismo , Eucariotos/genética , Desoxiadenosinas/genética , Mamíferos/metabolismoRESUMO
RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.
Assuntos
RNA , Transcrição Reversa , RNA/genética , RNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação/genética , Ligação ProteicaRESUMO
RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.
Assuntos
Aldeídos/química , RNA/química , Animais , Butanonas , Células-Tronco Embrionárias , Guanina/química , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes , Dobramento de RNA , TranscriptomaRESUMO
The gelsedine-type alkaloids possess a common oxabicyclo[3.2.2]nonane core and spiro- N-methoxyindolinone moiety along with a diversely functionalized heterocycle embedded in the compact framework. Herein we disclose a divergent entry to gelsedine-type alkaloids that hinges on rapid assembly of the common core by the orchestrated application of an asymmetric Michael addition, a tandem oxidation/aldol cyclization, and a pinacol rearrangement and generation of the structural diversity via a late-stage heterocyclization. The power of this strategy has been demonstrated through very short total syntheses of four gelsedine-type alkaloids: gelsedilam, gelsenicine, gelsedine, and gelsemoxonine.
RESUMO
Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.
Assuntos
Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Elementos Facilitadores Genéticos/genética , Cromatina/metabolismo , Cromatina/genética , Sítios de Ligação , Humanos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Transposases/metabolismo , Transposases/genética , Elementos Reguladores de Transcrição , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica , Diferenciação Celular/genética , Análise de Sequência de DNA/métodos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.
RESUMO
Fluxes in human copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate. We herein apply an unbiased temporal evaluation of the signaling and whole genome transcriptional activities modulated by copper level fluctuations to identify potential copper sensor proteins responsible for driving these activities. We find that fluctuations in physiologically relevant copper levels modulate EGFR signal transduction and activation of the transcription factor CREB. Both intracellular and extracellular assays support Cu1+ inhibition of the EGFR phosphatase PTPN2 (and potentially PTPN1)-via ligation to the PTPN2 active site cysteine side chain-as the underlying mechanism. We additionally show i) copper supplementation drives weak transcriptional repression of the copper importer CTR1 and ii) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper stimulated EGFR/CREB signaling and CTR1 expression.
Assuntos
Transportador de Cobre 1 , Cobre , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Receptores ErbB , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Transdução de Sinais , Receptores ErbB/metabolismo , Receptores ErbB/genética , Cobre/metabolismo , Humanos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transportador de Cobre 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.
Assuntos
Diferenciação Celular , Proliferação de Células , Metiltransferases , Neuroblastoma , Metiltransferases/metabolismo , Metiltransferases/antagonistas & inibidores , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Humanos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologiaRESUMO
N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m6A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233-129,263 sites using as little as 2 ng of poly A+ RNA. These identified sites correspond well with previous m6A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m6A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Perfilação da Expressão Gênica/métodos , RNA/genética , Biblioteca Gênica , Análise de Sequência de RNA/métodos , Mamíferos/genéticaRESUMO
Fluxes in human intra- and extracellular copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate (cAMP). We herein applied an unbiased temporal evaluation of the whole-genome transcriptional activities modulated by fluctuations in copper levels to identify the copper sensor proteins responsible for driving these activities. We found that fluctuations in physiologically-relevant copper levels rapidly modulate EGFR/MAPK/ERK signal transduction and activation of the transcription factor cAMP response element-binding protein (CREB). Both intracellular and extracellular assays support Cu 1+ inhibition of the EGFR-phosphatase PTPN2 (and potentially the homologous PTPN1)-via direct ligation to the PTPN2 active site cysteine side chain-as the underlying mechanism of copper-stimulated EGFR signal transduction activation. Depletion of copper represses this signaling pathway. We additionally show i ) copper supplementation drives transcriptional repression of the copper importer CTR1 and ii ) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper-stimulated MAPK/ERK/CREB-signaling and CTR1 expression, thereby uncovering a previously unrecognized link between copper levels and cellular signal transduction.
RESUMO
The first N-heterocyclic carbene-catalyzed stereoselective aza-Michael-Michael-lactonization cascade reaction of 2'-aminophenylenones and 2-bromoenals for the construction of chiral functionalized tetrahydroquinolines with three consecutive stereogenic centers has been achieved in high yields (up to 98%) with excellent diastereo- (>25:1) and enantioselectivities (up to 98.7% ee).