RESUMO
Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , CoesinasRESUMO
piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas de Caenorhabditis elegans/química , Quimera/metabolismo , Inativação Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.
Assuntos
Caenorhabditis elegans/metabolismo , Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Sequência de Aminoácidos , Animais , Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Exorribonucleases/química , Redes e Vias Metabólicas , Dados de Sequência Molecular , Mutação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , TranscriptomaRESUMO
Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.
Assuntos
Proteínas de Ciclo Celular , Cromátides , Humanos , Cromátides/genética , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , CoesinasRESUMO
Targeting epigenetic regulators to potentiate anti-PD-1 immunotherapy converges on the activation of type I interferon (IFN-I) response, mimicking cellular response to viral infection, but how its strength and duration are regulated to impact combination therapy efficacy remains largely unknown. Here, we show that mitochondrial CPT1A downregulation following viral infection restrains, while its induction by epigenetic perturbations sustains, a double-stranded RNA-activated IFN-I response. Mechanistically, CPT1A recruits the endoplasmic reticulum-localized ZDHHC4 to catalyze MAVS Cys79-palmitoylation, which promotes MAVS stabilization and activation by inhibiting K48- but facilitating K63-linked ubiquitination. Further elevation of CPT1A incrementally increases MAVS palmitoylation and amplifies the IFN-I response, which enhances control of viral infection and epigenetic perturbation-induced antitumor immunity. Moreover, CPT1A chemical inducers augment the therapeutic effect of combined epigenetic treatment with PD-1 blockade in refractory tumors. Our study identifies CPT1A as a stabilizer of MAVS activation, and its link to epigenetic perturbation can be exploited for cancer immunotherapy.
Assuntos
Interferon Tipo I , Viroses , Humanos , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lipoilação , Epigênese Genética , Imunidade InataRESUMO
In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4-7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin's loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Técnicas In Vitro , CoesinasRESUMO
Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Proteínas de Manutenção de Minicromossomo , Animais , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Fase G1 , Células HCT116 , Humanos , Camundongos , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação de Ácido Nucleico , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.
Assuntos
Cromátides , Proteínas Cromossômicas não Histona , Humanos , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mitose , DNA , Fase G2 , CoesinasRESUMO
Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.
Assuntos
Oryza , Tolerância ao Sal , Tolerância ao Sal/genética , Oryza/metabolismo , Peróxido de Hidrogênio/metabolismo , Homeostase , Dedos de Zinco , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/química , DNA/genética , Complexos Multiproteicos/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Quebra Cromossômica , Cromossomos/genética , DNA/química , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Conversão Gênica/genética , Células HCT116 , Humanos , Cinética , Complexos Multiproteicos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Torção Mecânica , Transcrição Gênica , Translocação Genética/genéticaRESUMO
Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Espermatozoides , Xenopus laevis , Animais , Masculino , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Espermatozoides/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Camundongos , Humanos , Cromatina/metabolismo , Cromatina/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Genoma , Núcleo Celular/metabolismoRESUMO
The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
Assuntos
Cromátides/química , Pareamento Cromossômico , Replicação do DNA , Genoma Humano/genética , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/análise , DNA/biossíntese , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , CoesinasRESUMO
Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Animais , Camundongos , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Mamíferos/genéticaRESUMO
Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal noncanonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNAâ¢ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of four putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in fragile X syndrome.
RESUMO
RNA methylation has emerged recently as an active research domain to study post-transcriptional alteration in gene expression regulation. Various types of RNA methylation, including N6-methyladenosine (m6A), are involved in human disease development. As a newly developed sequencing biotechnology to quantify the m6A level on a transcriptome-wide scale, MeRIP-seq expands RNA epigenetics study in both basic and clinical applications, with an upward trend. One of the fundamental questions in RNA methylation data analysis is to identify the Differentially Methylated Regions (DMRs), by contrasting cases and controls. Multiple statistical approaches have been recently developed for DMR detection, but there is a lack of a comprehensive evaluation for these analytical methods. Here, we thoroughly assess all eight existing methods for DMR calling, using both synthetic and real data. Our simulation adopts a Gamma-Poisson model and logit linear framework, and accommodates various sample sizes and DMR proportions for benchmarking. For all methods, low sensitivities are observed among regions with low input levels, but they can be drastically boosted by an increase in sample size. TRESS and exomePeak2 perform the best using metrics of detection precision, FDR, type I error control and runtime, though hampered by low sensitivity. DRME and exomePeak obtain high sensitivities, at the expense of inflated FDR and type I error. Analyses on three real datasets suggest differential preference on identified DMR length and uniquely discovered regions, between these methods.
Assuntos
RNA , Transcriptoma , Humanos , Análise de Sequência de RNA/métodos , RNA/genética , Metilação , Adenosina/genética , Adenosina/metabolismoRESUMO
Accounting for cell type compositions has been very successful at analyzing high-throughput data from heterogeneous tissues. Differential gene expression analysis at cell type level is becoming increasingly popular, yielding biomarker discovery in a finer granularity within a particular cell type. Although several computational methods have been developed to identify cell type-specific differentially expressed genes (csDEG) from RNA-seq data, a systematic evaluation is yet to be performed. Here, we thoroughly benchmark six recently published methods: CellDMC, CARseq, TOAST, LRCDE, CeDAR and TCA, together with two classical methods, csSAM and DESeq2, for a comprehensive comparison. We aim to systematically evaluate the performance of popular csDEG detection methods and provide guidance to researchers. In simulation studies, we benchmark available methods under various scenarios of baseline expression levels, sample sizes, cell type compositions, expression level alterations, technical noises and biological dispersions. Real data analyses of three large datasets on inflammatory bowel disease, lung cancer and autism provide evaluation in both the gene level and the pathway level. We find that csDEG calling is strongly affected by effect size, baseline expression level and cell type compositions. Results imply that csDEG discovery is a challenging task itself, with room to improvements on handling low signal-to-noise ratio and low expression genes.
Assuntos
Perfilação da Expressão Gênica , Software , Perfilação da Expressão Gênica/métodos , RNA-Seq , Simulação por Computador , Razão Sinal-Ruído , Análise de Sequência de RNA/métodosRESUMO
SUMMARY: Recent methodology advances in computational signal deconvolution have enabled bulk transcriptome data analysis at a finer cell-type level. Through deconvolution, identifying cell-type-specific differentially expressed (csDE) genes is drawing increasing attention in clinical applications. However, researchers still face a number of difficulties in adopting csDE genes detection methods in practice, especially in their experimental design. Here we present cypress, the first experimental design and statistical power analysis tool in csDE genes identification. This tool can reliably model purified cell-type-specific (CTS) profiles, cell-type compositions, biological and technical variations, offering a high-fidelity simulator for bulk RNA-seq convolution and deconvolution. cypress conducts simulation and evaluates the impact of multiple influencing factors, by various statistical metrics, to help researchers optimize experimental design and conduct power analysis. AVAILABILITY AND IMPLEMENTATION: cypress is an open-source R/Bioconductor package at https://bioconductor.org/packages/cypress/.
Assuntos
Perfilação da Expressão Gênica , Software , Humanos , Perfilação da Expressão Gênica/métodos , TranscriptomaRESUMO
Recently, novel biotechnologies to quantify RNA modifications became an increasingly popular choice for researchers who study epitranscriptome. When studying RNA methylations such as N6-methyladenosine (m6A), researchers need to make several decisions in its experimental design, especially the sample size and a proper statistical power. Due to the complexity and high-throughput nature of m6A sequencing measurements, methods for power calculation and study design are still currently unavailable. In this work, we propose a statistical power assessment tool, magpie, for power calculation and experimental design for epitranscriptome studies using m6A sequencing data. Our simulation-based power assessment tool will borrow information from real pilot data, and inspect various influential factors including sample size, sequencing depth, effect size, and basal expression ranges. We integrate two modules in magpie: (i) a flexible and realistic simulator module to synthesize m6A sequencing data based on real data; and (ii) a power assessment module to examine a set of comprehensive evaluation metrics.
Assuntos
Metilação de RNA , RNA , RNA/genética , RNA/metabolismo , Metilação , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Food fortification is an effective strategy to address vitamin A (VitA) deficiency, which is the leading cause of childhood blindness and drastically increases mortality from severe infections. However, VitA food fortification remains challenging due to significant degradation during storage and cooking. We utilized an FDA-approved, thermostable, and pH-responsive basic methacrylate copolymer (BMC) to encapsulate and stabilize VitA in microparticles (MPs). Encapsulation of VitA in VitA-BMC MPs greatly improved stability during simulated cooking conditions and long-term storage. VitA absorption was nine times greater from cooked MPs than from cooked free VitA in rats. In a randomized controlled cross-over study in healthy premenopausal women, VitA was readily released from MPs after consumption and had a similar absorption profile to free VitA. This VitA encapsulation technology will enable global food fortification strategies toward eliminating VitA deficiency.
Assuntos
Deficiência de Vitamina A , Vitamina A , Feminino , Ratos , Animais , Alimentos Fortificados , Estudos Cross-Over , Culinária , MicronutrientesRESUMO
BACKGROUND: The mediator complex subunits (MED) constitutes a multiprotein complex, with each subunit intricately involved in crucial aspects of plant growth, development, and responses to stress. Nevertheless, scant reports pertain to the VunMED gene within the context of asparagus bean (Vigna unguiculata ssp. sesquipedialis). Establishing the identification and exploring the responsiveness of VunMED to cold stress forms a robust foundation for the cultivation of cold-tolerant asparagus bean cultivars. RESULTS: Within this study, a comprehensive genome-wide identification of VunMED genes was executed in the asparagus bean cultivar 'Ningjiang3', resulting in the discovery of 36 distinct VunMED genes. A phylogenetic analysis encompassing 232 MED genes from diverse species, including Arabidopsis, tomatoes, soybeans, mung beans, cowpeas, and asparagus beans, underscored the highly conserved nature of MED gene sequences. Throughout evolutionary processes, each VunMED gene underwent purification and neutral selection, with the exception of VunMED19a. Notably, VunMED9/10b/12/13/17/23 exhibited structural variations discernible across four cowpea species. Divergent patterns of temporal and spatial expression were evident among VunMED genes, with a prominent role attributed to most genes during early fruit development. Additionally, an analysis of promoter cis-acting elements was performed, followed by qRT-PCR assessments on roots, stems, and leaves to gauge relative expression after exposure to cold stress and subsequent recovery. Both treatments induced transcriptional alterations in VunMED genes, with particularly pronounced effects observed in root-based genes following cold stress. Elucidating the interrelationships between subunits involved a preliminary understanding facilitated by correlation and principal component analyses. CONCLUSIONS: This study elucidates the pivotal contribution of VunMED genes to the growth, development, and response to cold stress in asparagus beans. Furthermore, it offers a valuable point of reference regarding the individual roles of MED subunits.