RESUMEN
Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.
Asunto(s)
Aminoacil-ARNt Sintetasas , Código Genético , Codón de Terminación/genética , Código Genético/genética , ARN de Transferencia/genética , Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genéticaRESUMEN
Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.
Asunto(s)
Aminoácidos , Sistemas CRISPR-Cas , Animales , Humanos , Codón de Terminación , Sistemas CRISPR-Cas/genética , Aminoácidos/química , Proteínas/metabolismo , Línea Celular , Mamíferos/genéticaRESUMEN
DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape's molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.
Asunto(s)
Epigénesis Genética , Epigenoma , Animales , Ratones , Femenino , Embarazo , Epigenoma/genética , Placenta/metabolismo , Metilación de ADN , Proteínas del Grupo Polycomb/genética , ADN/metabolismo , Mamíferos/metabolismoRESUMEN
DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/metabolismo , Animales , Citosina/metabolismo , Desmetilación del ADN , Metilación de ADN , Proteínas de Unión al ADN/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Expresión Génica , Mamíferos/genética , Ratones , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an 'activated' state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.
Asunto(s)
Células Madre Embrionarias Humanas , Complejo Represivo Polycomb 2 , Diferenciación Celular/genética , Desarrollo Embrionario , Histonas/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.
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Daño del ADN , Neoplasias , Cromatina , ADN/efectos de la radiación , Roturas del ADN de Doble Cadena , Reparación del ADN , Neoplasias/genéticaRESUMEN
Trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.
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Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Lisina , Sirtuina 1 , Acetilación , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Lisina/metabolismo , Agregación Patológica de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT-seq) to define both coding and non-coding transcription units (TUs) in these three pluripotent states and combine TT-seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome-wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome-wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome-wide changes in transcription kinetics invoke ground and paused pluripotent states.
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ARN Polimerasa II , Transcriptoma , Animales , Cinética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (ESC), observing widespread G4 formation at active promoters, active and poised enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mouse ESCs. Upon differentiation to neural progenitor cells (NPC), enhancer G4s are lost. Further, performing R-loop CUT&Tag, we demonstrate the genome-wide co-occurrence of single-stranded DNA, G4s and R loops at promoters and enhancers. We confirm that G4 structures exist independent of ongoing transcription, suggesting an intricate relationship between transcription and non-canonical DNA structures.
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G-Cuádruplex , Animales , ADN/química , ADN/genética , Inestabilidad Genómica , Mamíferos , Ratones , Estructuras R-Loop , ARNRESUMEN
Short ORFs (sORFs), that is, occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding ORFs by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural, and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization.
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Sistemas de Lectura Abierta/genética , Proteínas/genética , Ribosomas/genética , Codón Iniciador/genética , Codón de Terminación/genética , Biología Computacional , Anotación de Secuencia MolecularRESUMEN
Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.
RESUMEN
This paper aims to help policy makers with a characterization of the intrinsic value of biodiversity and its role as a critical foundation for sustainable development, human health, and well-being. Our objective is to highlight the urgent need to overcome economic, disciplinary, national, cultural, and regional barriers, in order to work out innovative measures to create a sustainable future and prevent the mutual extinction of humans and other species. We emphasize the pervasive neglect paid to the cross-dependency of planetary health, the health of individual human beings and other species. It is critical that social and natural sciences are taken into account as key contributors to forming policies related to biodiversity, conservation, and health management. We are reaching the target date of Nagoya treaty signatories to have accomplished measures to prevent biodiversity loss, providing a unique opportunity for policy makers to make necessary adjustments and refocus targets for the next decade. We propose recommendations for policy makers to explore novel avenues to halt the accelerated global loss of biodiversity. Beyond the critical ecological functions biodiversity performs, its enormous untapped the repertoire of natural molecular diversity is needed for solving accelerating global healthcare challenges.
Asunto(s)
Biodiversidad , Descubrimiento de Drogas/métodos , Política de Salud/tendencias , Desarrollo Sostenible/tendencias , Descubrimiento de Drogas/normas , HumanosRESUMEN
Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.
Asunto(s)
ARN Helicasas DEAD-box , Proteína p53 Supresora de Tumor , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Exones/genética , Ratones , Ribosomas/genética , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models.
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Aminoácidos/metabolismo , Encéfalo/metabolismo , Ingeniería Celular , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , Aminoácidos/genética , Código Genético , HumanosRESUMEN
RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.
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COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virología , Calor , Humanos , Nasofaringe/virología , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Juego de Reactivos para Diagnóstico , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Inactivación de VirusRESUMEN
Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.
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Colorantes Fluorescentes/química , Proteínas/química , Animales , Células HEK293 , Humanos , Microscopía Fluorescente , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. Nucleosome turnover is known to be universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently at intracisternal A-particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28-KAP1/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that Smarcad1 chromatin remodeler evicts nucleosomes generating accessible DNA. Free DNA is repackaged via DAXX-mediated nucleosome assembly with histone variant H3.3 in this dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.
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Células Madre Embrionarias/fisiología , Heterocromatina/metabolismo , Histonas/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , ADN/metabolismo , ADN Helicasas/metabolismo , Heterocromatina/genética , Histonas/genética , Ratones Noqueados , Nucleosomas/genética , Nucleosomas/metabolismo , Células Madre Pluripotentes/fisiología , Retroelementos/genéticaRESUMEN
The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from the archaeon Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the G1 PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation of two lysine analogs with high selectivity and efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G protein-coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells.
Asunto(s)
Aminoácidos/metabolismo , Reactivos de Enlaces Cruzados/química , Mamíferos/metabolismo , Proteínas/metabolismo , Coloración y Etiquetado , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Secuencia de Bases , Línea Celular , Supervivencia Celular , Células HEK293 , Humanos , Lisina/metabolismo , Methanosarcina/metabolismo , Conformación de Ácido Nucleico , ARN de Transferencia/química , ARN de Transferencia/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Especificidad por SustratoRESUMEN
RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.