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1.
Int J Mol Sci ; 24(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36614240

RESUMO

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.


Assuntos
Frutas , Vitis , Frutas/genética , Óvulo Vegetal/genética , RNA/metabolismo , Endopeptidase K/metabolismo , Sementes/metabolismo , Vitis/genética
2.
Int J Mol Sci ; 24(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36614310

RESUMO

Exosomes may function as multifactorial mediators of cell-to-cell communication, playing crucial roles in both physiological and pathological processes. Exosomes released from virus-infected cells may contain RNA and proteins facilitating infection spread. The purpose of our study was to analyze how the small RNA content of exosomes is affected by infection with the influenza A virus (IAV). Exosomes were isolated by ultracentrifugation after hemadsorption of virions and their small RNA content was identified using high-throughput sequencing. As compared to mock-infected controls, 856 RNA transcripts were significantly differentially expressed in exosomes from IAV-infected cells, including fragments of 458 protein-coding (pcRNA), 336 small, 28 long intergenic non-coding RNA transcripts, and 33 pseudogene transcripts. Upregulated pcRNA species corresponded mainly to proteins associated with translation and antiviral response, and the most upregulated among them were RSAD2, CCDC141 and IFIT2. Downregulated pcRNA species corresponded to proteins associated with the cell cycle and DNA packaging. Analysis of differentially expressed pseudogenes showed that in most cases, an increase in the transcription level of pseudogenes was correlated with an increase in their parental genes. Although the role of exosome RNA in IAV infection remains undefined, the biological processes identified based on the corresponding proteins may indicate the roles of some of its parts in IAV replication.


Assuntos
Exossomos , Vírus da Influenza A , Influenza Humana , Humanos , RNA/metabolismo , Vírus da Influenza A/genética , Exossomos/genética , Células Epiteliais/metabolismo , Proteínas/metabolismo , Replicação Viral
3.
Molecules ; 28(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36677577

RESUMO

(1) Background: Icariin is the main component of the Chinese herb Epimedium. A number of studies have shown that it alleviates abnormal lipid metabolism. However, it is not clear whether and how icariin can ameliorate hepatic steatosis with polycystic ovary syndrome (PCOS). This study was designed to explore the anti-hepatosteatosis effect of icariin in rats with polycystic ovary syndrome. (2) Methods: Female Sprague Dawley(SD)rats were treated with a high-fat diet and letrozole for 21 days to make nonalcoholic fatty liver disease (NAFLD) in the polycystic ovary syndrome model. Then model rats were treated with icariin (by gavage, once daily) for 28 days. Serum hormones and biochemical variables were determined by ELISA or enzyme. RNA-sequence analysis was used to enrich related target pathways. Then, quantitative Real-time PCR (qRT-PCR) and Western blot were performed to verify target genes and proteins. (3) Results: Icariin treatment reduced excess serum levels of Testosterone (T), Estradiol (E2), Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), LH/FSH ratio, insulin, triglycerides (TG), and aspartate aminotransferase (AST) in high-fat diet (HFD) and letrozole fed rats. Meanwhile, icariin ameliorated HFD and letrozole-induced fatty liver, as evidenced by a reduction in excess triglyceride accumulation, vacuolization, and Oil Red O staining area in the liver of model rats. Results of RNA-sequencing, western blotting, and qRT-PCR analyses indicated that icariin up-regulated fatty acid translocase (CD36), in mitochondria, and peroxisome proliferator-activated receptor α (PPARα) expression, which led to the enhancement of fatty acid oxidation molecules, such as cytochrome P450, family 4, subfamily a, polypeptide 3 (CYP4A3), carnitine palmitoyltransferase 1 α (CPT1α), acyl-CoA oxidase 1 (ACOX1), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD). Besides, icariin reduced lipid synthesis, which elicited stearoyl-Coenzyme A desaturase 1 (SCD1), fatty acid synthase (FASN), and acetyl-CoA (ACC). (4) Conclusion: Icariin showed an ameliorative effect on hepatic steatosis induced by HFD and letrozole, which was associated with improved fatty acid oxidation and reduced lipid accumulation in the liver.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Síndrome do Ovário Policístico , Feminino , Humanos , Ratos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Letrozol/farmacologia , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Ratos Sprague-Dawley , Fígado , Metabolismo dos Lipídeos , Dieta Hiperlipídica/efeitos adversos , Triglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Hormônio Foliculoestimulante/metabolismo , RNA/metabolismo
4.
Proc Natl Acad Sci U S A ; 120(4): e2216436120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36656865

RESUMO

Enhancers not only activate target promoters to stimulate messenger RNA (mRNA) synthesis, but they themselves also undergo transcription to produce enhancer RNAs (eRNAs), the significance of which is not well understood. Transcription at the participating enhancer-promoter pair appears coordinated, but it is unclear why and how. Here, we employ cell-free transcription assays using constructs derived from the human GREB1 locus to demonstrate that transcription at an enhancer and its target promoter is interdependent. This interdependence is observable under conditions where direct enhancer-promoter contact (EPC) takes place. We demonstrate that transcription activation at a participating enhancer-promoter pair is dependent on i) the mutual availability of the enhancer and promoter, ii) the state of transcription at both the enhancer and promoter, iii) local abundance of both eRNA and mRNA, and iv) direct EPC. Our results suggest transcriptional interdependence between the enhancer and the promoter as the basis of their transcriptional concurrence and coordination throughout the genome. We propose a model where transcriptional concurrence, coordination and interdependence are possible if the participating enhancer and promoter are entangled in the form of EPC, reside in a proteinaceous bubble, and utilize shared transcriptional resources and regulatory inputs.


Assuntos
Elementos Facilitadores Genéticos , RNA , Humanos , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , RNA/genética , RNA Mensageiro/genética , Ativação Transcricional , Transcrição Genética , Regulação da Expressão Gênica
5.
J Acquir Immune Defic Syndr ; 92(2): 180-188, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36625858

RESUMO

BACKGROUND: We evaluated trends in CD4/CD8 ratio among people living with HIV (PLWH) starting antiretroviral therapy (ART) with first-line integrase strand transfer inhibitors (INSTI) compared with non-INSTI-based ART, and the incidence of CD4/CD8 ratio normalization. METHODS: All PLWH enrolled in adult HIV cohorts of IeDEA Asia-Pacific who started with triple-ART with at least 1 CD4, CD8 (3-month window), and HIV-1 RNA measurement post-ART were included. CD4/CD8 ratio normalization was defined as a ratio ≥1. Longitudinal changes in CD4/CD8 ratio were analyzed by linear mixed model, the incidence of the normalization by Cox regression, and the differences in ratio recovery by group-based trajectory modeling. RESULTS: A total of 5529 PLWH were included; 80% male, median age 35 years (interquartile range [IQR], 29-43). First-line regimens were comprised of 65% NNRTI, 19% PI, and 16% INSTI. The baseline CD4/CD8 ratio was 0.19 (IQR, 0.09-0.33). PLWH starting with NNRTI- (P = 0.005) or PI-based ART (P = 0.030) had lower CD4/CD8 recovery over 5 years compared with INSTI. During 24,304 person-years of follow-up, 32% had CD4/CD8 ratio normalization. After adjusting for age, sex, baseline CD4, HIV-1 RNA, HCV, and year of ART initiation, PLWH started with INSTI had higher odds of achieving CD4/CD8 ratio normalization than NNRTI- (P < 0.001) or PI-based ART (P = 0.015). In group-based trajectory modeling analysis, INSTI was associated with greater odds of being in the higher ratio trajectory. CONCLUSIONS: INSTI use was associated with higher rates of CD4/CD8 ratio recovery and normalization in our cohort. These results emphasize the relative benefits of INSTI-based ART for immune restoration.


Assuntos
Infecções por HIV , Inibidores de Integrase de HIV , Adulto , Humanos , Masculino , Feminino , Infecções por HIV/tratamento farmacológico , Estudos Prospectivos , Estudos de Coortes , Relação CD4-CD8 , Inibidores de Integrase de HIV/uso terapêutico , Linfócitos T CD8-Positivos , RNA/uso terapêutico , Integrases
6.
Nat Commun ; 14(1): 205, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36639366

RESUMO

Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. In this report, we showed that the folding of putative rG4s in human cells into rG4 structures is dynamically regulated under stress. By using high-throughput dimethylsulfate (DMS) probing, we identified hundreds of endogenous stress-induced rG4s, and validated them by using an rG4 pull-down approach. Our results demonstrate that stress-induced rG4s are enriched in mRNA 3'-untranslated regions and enhance mRNA stability. Furthermore, stress-induced rG4 folding is readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in human cells and suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.


Assuntos
Quadruplex G , RNA , Humanos , RNA/genética , RNA/química , RNA Mensageiro/genética , Dobramento de RNA , Estabilidade de RNA
7.
BMC Neurosci ; 24(1): 1, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36604634

RESUMO

BACKGROUND: The role of cytokines in the pathophysiology, diagnosis, and prognosis of small fiber neuropathy (SFN) is incompletely understood. We studied expression profiles of selected pro- and anti-inflammatory cytokines in RNA from white blood cells (WBC) of patients with a medical history and a clinical phenotype suggestive for SFN and compared data with healthy controls. METHODS: We prospectively recruited 52 patients and 21 age- and sex-matched healthy controls. Study participants were characterized in detail and underwent complete neurological examination. Venous blood was drawn for routine and extended laboratory tests, and for WBC isolation. Systemic RNA expression profiles of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-2, IL-8, tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4, IL-10, transforming growth factor beta-1 (TGF) were analyzed. Protein levels of IL-2, IL-8, and TNF were measured in serum of patients and controls. Receiver operating characteristic (ROC)-curve analysis was used to determine the accuracy of IL-2, IL-8, and TNF in differentiating patients and controls. To compare the potential discriminatory efficacy of single versus combined cytokines, equality of different AUCs was tested. RESULTS: WBC gene expression of IL-2, IL-8, and TNF was higher in patients compared to healthy controls (IL-2: p = 0.02; IL-8: p = 0.009; TNF: p = 0.03) and discriminated between the groups (area under the curve (AUC) ≥ 0.68 for each cytokine) with highest diagnostic accuracy reached by combining the three cytokines (AUC = 0.81, sensitivity = 70%, specificity = 86%). Subgroup analysis revealed the following differences: IL-8 and TNF gene expression levels were higher in female patients compared to female controls (IL-8: p = 0.01; TNF: p = 0.03). The combination of TNF with IL-2 and TNF with IL-2 and IL-8 discriminated best between the study groups. IL-2 was higher expressed in patients with moderate pain compared to those with severe pain (p = 0.02). Patients with acral pain showed higher IL-10 gene expression compared to patients with generalized pain (p = 0.004). We further found a negative correlation between the relative gene expression of IL-2 and current pain intensity (p = 0.02). Serum protein levels of IL-2, IL-8, and TNF did not differ between patients and controls. CONCLUSIONS: We identified higher systemic gene expression of IL-2, IL-8, and TNF in SFN patients than in controls, which may be of potential relevance for diagnostics and patient stratification.


Assuntos
Citocinas , Neuropatia de Pequenas Fibras , Feminino , Humanos , Interleucina-10 , Interleucina-2 , Interleucina-8 , Leucócitos/química , Dor , RNA , Fator de Necrose Tumoral alfa
8.
Sci Rep ; 13(1): 350, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611052

RESUMO

In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein-protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues' interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14's exoribonuclease (ExoN) and nsp16's 2'O-methyltransferase (2'O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2'O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell's innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein-protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide-protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Simulação de Acoplamento Molecular , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Replicação Viral/genética , Metiltransferases/genética , Peptídeos/farmacologia , Antivirais/farmacologia , RNA/farmacologia , Exorribonucleases/genética , Exorribonucleases/química
9.
BMC Plant Biol ; 23(1): 31, 2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36639742

RESUMO

BACKGROUND: Auxin performs important functions in plant growth and development processes, as well as abiotic stress. Small auxin-up RNA (SAUR) is the largest gene family of auxin-responsive factors. However, the knowledge of the SAUR gene family in foxtail millet is largely obscure. RESULTS: In the current study, 72 SiSAUR genes were identified and renamed according to their chromosomal distribution in the foxtail millet genome. These SiSAUR genes were unevenly distributed on nine chromosomes and were classified into three groups through phylogenetic tree analysis. Most of the SiSAUR members from the same group showed similar gene structure and motif composition characteristics. Analysis of cis-acting elements showed that many hormone and stress response elements were identified in the promoter region of SiSAURs. Gene replication analysis revealed that many SiSAUR genes were derived from gene duplication events. We also found that the expression of 10 SiSAURs was induced by abiotic stress and exogenous hormones, which indicated that SiSAUR genes may participated in complex physiological processes. CONCLUSIONS: Overall, these results will be valuable for further studies on the biological role of SAUR genes in foxtail development and response to stress conditions and may shed light on the improvement of the genetic breeding of foxtail millet.


Assuntos
Setaria (Planta) , Setaria (Planta)/genética , Setaria (Planta)/metabolismo , Ácidos Indolacéticos/metabolismo , RNA/metabolismo , Família Multigênica , Filogenia , Melhoramento Vegetal , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo
10.
Nat Commun ; 14(1): 332, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658112

RESUMO

RNA molecules aggregate under certain conditions. The resulting condensates are implicated in human neurological disorders, and can potentially be designed towards specified bulk properties in vitro. However, the mechanism for aggregation-including how aggregation properties change with sequence and environmental conditions-remains poorly understood. To address this challenge, we introduce an analytical framework based on multimer enumeration. Our approach reveals the driving force for aggregation to be the increased configurational entropy associated with the multiplicity of ways to form bonds in the aggregate. Our model uncovers rich phase behavior, including a sequence-dependent reentrant phase transition, and repeat parity-dependent aggregation. We validate our results by comparison to a complete computational enumeration of the landscape, and to previously published molecular dynamics simulations. Our work unifies and extends published results, both explaining the behavior of CAG-repeat RNA aggregates implicated in Huntington's disease, and enabling the rational design of programmable RNA condensates.


Assuntos
Doença de Huntington , RNA , Gravidez , Feminino , Humanos , RNA/genética , Doença de Huntington/genética
11.
Nat Commun ; 14(1): 334, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658122

RESUMO

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand.


Assuntos
Pseudouridina , Saccharomyces cerevisiae , Animais , Humanos , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , RNA , Transcriptoma , Mamíferos/genética
12.
Nat Commun ; 14(1): 315, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658155

RESUMO

N6-methyladenosine (m6A) has been demonstrated to regulate RNA metabolism and various biological processes, including gametogenesis and embryogenesis. However, the landscape and function of m6A at single cell resolution have not been extensively studied in mammalian oocytes or during pre-implantation. In this study, we developed a single-cell m6A sequencing (scm6A-seq) method to simultaneously profile the m6A methylome and transcriptome in single oocytes/blastomeres of cleavage-stage embryos. We found that m6A deficiency leads to aberrant RNA clearance and consequent low quality of Mettl3Gdf9 conditional knockout (cKO) oocytes. We further revealed that m6A regulates the translation and stability of modified RNAs in metaphase II (MII) oocytes and during oocyte-to-embryo transition, respectively. Moreover, we observed m6A-dependent asymmetries in the epi-transcriptome between the blastomeres of two-cell embryo. scm6A-seq thus allows in-depth investigation into m6A characteristics and functions, and the findings provide invaluable single-cell resolution resources for delineating the underlying mechanism for gametogenesis and early embryonic development.


Assuntos
Oócitos , Oogênese , Animais , Oócitos/metabolismo , Desenvolvimento Embrionário/genética , Transcriptoma/genética , RNA/metabolismo , Mamíferos/genética
13.
Genome Biol ; 24(1): 15, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658633

RESUMO

BACKGROUND: Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export. RESULTS: We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression. CONCLUSIONS: This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.


Assuntos
RNA , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Transporte Ativo do Núcleo Celular , RNA/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675253

RESUMO

Over the years, increasing evidence has shown that copy number variations (CNVs) play an important role in the pathogenesis and prognosis of Colorectal Cancer (CRC). Colorectal adenomas are highly prevalent lesions, but only 5% of these adenomas ever progress to carcinoma. This review summarizes the different CNVs associated with adenoma-carcinoma CRC progression and with CRC staging. Characterization of CNVs in circulating free-RNA and in blood-derived exosomes augers well with the potential of using such assays for patient management and early detection of metastasis. To overcome the limitations related to tissue biopsies and tumor heterogeneity, using CNVs to characterize tumor-derived materials in biofluids provides less invasive sampling methods and a sample that collectively represents multiple tumor sites in heterogeneous samples. Liquid biopsies provide a source of circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), tumor-derived exosomes (TDE), circulating free RNA, and non-coding RNA. This review provides an overview of the current diagnostic and predictive models from liquid biopsies.


Assuntos
Adenoma , Carcinoma , Ácidos Nucleicos Livres , Neoplasias Colorretais , Células Neoplásicas Circulantes , Humanos , Variações do Número de Cópias de DNA/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Biópsia Líquida/métodos , Ácidos Nucleicos Livres/genética , Células Neoplásicas Circulantes/patologia , RNA , Biomarcadores Tumorais/genética , Adenoma/diagnóstico , Adenoma/genética
15.
Molecules ; 28(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36677900

RESUMO

Despite structural differences between the right-handed conformations of A-RNA and B-DNA, both nucleic acids adopt very similar, left-handed Z-conformations. In contrast to their structural similarities and sequence preferences, RNA and DNA exhibit differences in their ability to adopt the Z-conformation regarding their hydration shells, the chemical modifications that promote the Z-conformation, and the structure of junctions connecting them to right-handed segments. In this review, we highlight the structural and chemical properties of both Z-DNA and Z-RNA and delve into the potential factors that contribute to both their similarities and differences. While Z-DNA has been extensively studied, there is a gap of knowledge when it comes to Z-RNA. Where such information is lacking, we try and extend the principles of Z-DNA stability and formation to Z-RNA, considering the inherent differences of the nucleic acids.


Assuntos
DNA Forma Z , Ácidos Nucleicos , RNA , Conformação de Ácido Nucleico , DNA/química
16.
Commun Biol ; 6(1): 82, 2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681772

RESUMO

RNA-binding proteins (RBPs) are key players of gene expression and perturbations of RBP-RNA regulatory network have been observed in various cancer types. Here, we propose a computational method, RBPreg, to identify the RBP regulators by integration of single cell RNA-Seq (N = 233,591) and RBP binding data. Pan-cancer analyses suggest that RBP regulators exhibit cancer and cell specificity and perturbations of RBP regulatory network are involved in cancer hallmark-related functions. We prioritize an oncogenic RBP-HNRNPK, which is highly expressed in tumors and associated with poor prognosis of patients. Functional assays performed in cancer cells reveal that HNRNPK promotes cancer cell proliferation, migration, and invasion in vitro and in vivo. Mechanistic investigations further demonstrate that HNRNPK promotes tumorigenesis and progression by directly binding to MYC and perturbed the MYC targets pathway in lung cancer. Our results provide a valuable resource for characterizing RBP regulatory networks in cancer, yielding potential biomarkers for precision medicine.


Assuntos
Neoplasias Pulmonares , RNA , Humanos , RNA/genética , Carcinogênese , Transformação Celular Neoplásica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética
17.
Lett Appl Microbiol ; 76(1)2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36688775

RESUMO

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32 min), although the best conditions were 38°C for 30 min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.


Assuntos
Citrus , Transcrição Reversa , Recombinases/metabolismo , Ácido Edético , Hidróxido de Sódio , RNA , Citrus/metabolismo , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido Nucleico
18.
Methods Mol Biol ; 2630: 103-115, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36689179

RESUMO

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.


Assuntos
Pequeno RNA não Traduzido , RNA , Animais , Biblioteca Gênica , Oligonucleotídeos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Clonagem Molecular , Análise de Sequência de RNA/métodos , Pequeno RNA não Traduzido/genética
19.
Commun Biol ; 6(1): 93, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690686

RESUMO

Mitochondrial DNA (mtDNA) harbors essential genes in most metazoans, yet the regulatory impact of the multiple evolutionary mtDNA rearrangements has been overlooked. Here, by analyzing mtDNAs from ~8000 metazoans we found high gene content conservation (especially of protein and rRNA genes), and codon preferences for mtDNA-encoded tRNAs across most metazoans. In contrast, mtDNA gene order (MGO) was selectively constrained within but not between phyla, yet certain gene stretches (ATP8-ATP6, ND4-ND4L) were highly conserved across metazoans. Since certain metazoans with different MGOs diverge in mtDNA transcription, we hypothesized that evolutionary mtDNA rearrangements affected mtDNA transcriptional patterns. As a first step to test this hypothesis, we analyzed available RNA-seq data from 53 metazoans. Since polycistron mtDNA transcripts constitute a small fraction of the steady-state RNA, we enriched for polycistronic boundaries by calculating RNA-seq read densities across junctions between gene couples encoded either by the same strand (SSJ) or by different strands (DSJ). We found that organisms whose mtDNA is organized in alternating reverse-strand/forward-strand gene blocks (mostly arthropods), displayed significantly reduced DSJ read counts, in contrast to organisms whose mtDNA genes are preferentially encoded by one strand (all chordates). Our findings suggest that mtDNA rearrangements are selectively constrained and likely impact mtDNA regulation.


Assuntos
DNA Mitocondrial , Mitocôndrias , Animais , DNA Mitocondrial/genética , Ordem dos Genes , Mitocôndrias/metabolismo , RNA/metabolismo , RNA de Transferência/genética
20.
J Transl Med ; 21(1): 41, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36691026

RESUMO

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity. METHODS: We analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results. RESULTS: Although panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes. CONCLUSION: Our research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Humanos , Neoplasias Colorretais/genética , Proteômica/métodos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Medicina de Precisão , Neoplasias Hepáticas/genética , RNA , Biomarcadores Tumorais , Fatores de Processamento de Serina-Arginina
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