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1.
BMC Cancer ; 22(1): 857, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35931993

RESUMO

BACKGROUND: Liver cirrhosis is a well-known risk factor for hepatocellular carcinoma (HCC). However, some HCC cases can also originate from non-cirrhotic livers. The aim of this study was to identify key circular RNAs (circRNAs) associated with the tumorigenesis of non-cirrhotic liver disease. METHODS: The differently expressed circRNAs between non-cirrhotic and cirrhotic HCCs were assessed with use of high-throughput circRNAs sequencing and validated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Potential biological functions of these dysregulated circRNAs were predicted with use of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA-miRNA-mRNA regulation network was constructed as achieved with use of miRanda software and visualized using Cytoscape software. Biological functions of the four most prominent dysregulated circRNAs identified were confirmed by in vitro experiments. Moreover, possible translations of these dysregulated circRNAs were also predicted. RESULTS: A total of 393 dysregulated circRNAs were identified between non-cirrhotic and cirrhotic HCC, including 213 that were significantly up-regulated and 180 significantly down-regulated circRNAs. Expression levels of the six most prominent dysregulated circRNAs were further validated using qRT-PCR. Many tumor related miRNAs were involved in the circRNA-miRNA-mRNA networks, including miR-182-5p, miR-561-3p, miR-125a-5p, miR-145, miR-23b-3p and miR-30e-3p, and downstream mRNAs of dysregulated circRNAs were significantly related with biological processes involved in the progression of tumors, including proliferation, migration, differentiation, and focal adhesion. Results from the in vitro experiments demonstrated that the most prominent dysregulated circRNAs exerted notable effects upon the proliferation and migration of HCC cells. Finally, we also identified 19 dysregulated circRNAs having potential for the coding of functional peptides. CONCLUSION: The results of this present study indicate that circRNAs may play important roles in tumorigenesis of non-cirrhotic HCC. Such findings provide some novel insights and pave the way for the development of future studies directed at investigating the initiation and treatment of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Carcinogênese , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/genética , RNA/metabolismo , RNA Circular/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
2.
Genome Biol ; 23(1): 167, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927734

RESUMO

BACKGROUND: Plants undergo programmed chromatin changes in response to environment, influencing heritable phenotypic plasticity. The RNA-directed DNA methylation (RdDM) pathway is an essential component of this reprogramming process. The relationship of epigenomic changes to gene networks on a genome-wide basis has been elusive, particularly for intragenic DNA methylation repatterning. RESULTS: Epigenomic reprogramming is tractable to detailed study and cross-species modeling in the MSH1 system, where perturbation of the plant-specific gene MSH1 triggers at least four distinct nongenetic states to impact plant stress response and growth vigor. Within this system, we have defined RdDM target loci toward decoding phenotype-relevant methylome data. We analyze intragenic methylome repatterning associated with phenotype transitions, identifying state-specific cytosine methylation changes in pivotal growth-versus-stress, chromatin remodeling, and RNA spliceosome gene networks that encompass 871 genes. Over 77% of these genes, and 81% of their central network hubs, are functionally confirmed as RdDM targets based on analysis of mutant datasets and sRNA cluster associations. These dcl2/dcl3/dcl4-sensitive gene methylation sites, many present as singular cytosines, reside within identifiable sequence motifs. These data reflect intragenic methylation repatterning that is targeted and amenable to prediction. CONCLUSIONS: A prevailing assumption that biologically relevant DNA methylation variation occurs predominantly in density-defined differentially methylated regions overlooks behavioral features of intragenic, single-site cytosine methylation variation. RdDM-dependent methylation changes within identifiable sequence motifs reveal gene hubs within networks discriminating stress response and growth vigor epigenetic phenotypes. This study uncovers components of a methylome "code" for de novo intragenic methylation repatterning during plant phenotype transitions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , Metilação de DNA , Epigênese Genética , Epigenoma , Regulação da Expressão Gênica de Plantas , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , RNA/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/genética
3.
Genome Biol ; 23(1): 169, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927743

RESUMO

BACKGROUND: Adenosine-to-inosine (A-to-I) editing is an important RNA posttranscriptional process related to a multitude of cellular and molecular activities. However, systematic characterizations of whether and how the events of RNA editing are associated with the binding preferences of RNA sequences to RNA-binding proteins (RBPs) are still lacking. RESULTS: With the RNA-seq and RBP eCLIP-seq datasets from the ENCODE project, we quantitatively survey the binding preferences of 150 RBPs to RNA editing events, followed by experimental validations. Such analyses of the RBP-associated RNA editing at nucleotide resolution and genome-wide scale shed light on the involvement of RBPs specifically in RNA editing-related processes, such as RNA splicing, RNA secondary structures, RNA decay, and other posttranscriptional processes. CONCLUSIONS: These results highlight the relevance of RNA editing in the functions of many RBPs and therefore serve as a resource for further characterization of the functional associations between various RNA editing events and RBPs.


Assuntos
Edição de RNA , Proteínas de Ligação a RNA , Genoma , RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo
4.
J Med Chem ; 65(15): 10217-10232, 2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-35916496

RESUMO

Targeted protein degradation (TPD), represented by proteolysis-targeting chimera (PROTAC), has emerged as a novel therapeutic modality in drug discovery. However, the application of conventional PROTACs is limited to protein targets containing cytosolic domains with ligandable sites. Recently, nucleic-acid-based modalities, such as modified oligonucleotide mimics and aptamers, opened new avenues to degrade protein targets and greatly expanded the scope of TPD. Beyond constructing protein-degrading chimeras, nucleic acid motifs can also serve as substrates for targeted degradation. Particularly, the new type of chimeric RNA degrader termed ribonuclease-targeting chimera (RIBOTAC) has shown promising features in drug discovery. Here, we provide an overview of the newly emerging TPD strategies based on nucleic acids as well as new strategies for targeted degradation of nucleic acid (RNA) targets. The design strategies, case studies, potential applications, and challenges are focused on.


Assuntos
Ácidos Nucleicos , Quimera/metabolismo , Descoberta de Drogas , Proteínas/metabolismo , Proteólise , RNA/metabolismo
5.
Front Immunol ; 13: 905211, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35936005

RESUMO

N6-metyladenosine (m6A) RNA methylation has been proven to be involved in diverse biological processes, but its potential roles in the development of lipopolysaccharide (LPS) induced retinal pigment epithelium (RPE) inflammation have not been revealed. In this study, we explored the effects and underlying mechanisms of methyltransferase-like 3 (METTL3) in LPS stimulated RPE cells. Proliferation of METTL3-silenced RPE cells was examined by Cell counting kit-8 (CCK8) and 5-Ethynyl-2´-Deoxyuridine (Edu). Expression of tight junction proteins ZO-1 and Occludin, and secretion of inflammatory factors interleukins (IL)-1, 6 and 8 were detected by Western blotting or Enzyme-linked immunosorbent assay (ELISA). RNA sequencing and methylated RNA immunoprecipitation (MeRIP) sequencing were used to analyze the target gene nuclear receptor subfamily 2 group F member 1 (NR2F1) of METTL3. Our results showed that both human RPE (hRPE) cells and ARPE19 cells exhibited inhibited proliferation, tight junction protein expression, and increased inflammatory factor secretion after METTL3 silencing. Mechanistically, we found that NR2F1, as a METTL3-methylated target gene, inhibits Occludin level and promotes IL-6 secretion of RPE cells in an m6A-dependent manner. Interestingly, NR2F1 deficiency reversed the decreased Occludin expression and increased IL-6 secretion in METTL3-defective RPE cells. In conclusion, our study revealed that METTL3 attenuates RPE cell inflammation by methylating NR2F1, suggesting the critical role of METTL3 in RPE cells.


Assuntos
Fator I de Transcrição COUP/metabolismo , Lipopolissacarídeos , Metiltransferases/metabolismo , Epitélio Pigmentado da Retina , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ocludina/metabolismo , RNA/metabolismo , Epitélio Pigmentado da Retina/metabolismo
6.
Nat Commun ; 13(1): 4624, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941106

RESUMO

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.


Assuntos
Proteínas Argonauta , RNA , Proteínas Argonauta/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Células Procarióticas/metabolismo , RNA/metabolismo , RNA Guia/genética , RNA Guia/metabolismo
7.
Life Sci Alliance ; 5(12)2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35914813

RESUMO

Over the last decades, organoids have been established from most of the tissue-resident stem and iPS cells. They hold great promise for our understanding of mammalian organ development, but also for the study of disease or even personalised medicine. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such heterogeneity has not been performed before. Here, we used RNA-seq of individual intrahepatic cholangiocyte organoids to address this question. We find that batch-to-batch variation is very low, whereas passage number has a profound impact on gene expression profiles. On the other hand, there is organoid-to-organoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Taken together, our study provides a framework for organoid researchers to properly consider experimental design.


Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Animais , Células Epiteliais , Mamíferos , Organoides/metabolismo , RNA/metabolismo , Análise de Sequência de RNA
8.
Proc Natl Acad Sci U S A ; 119(33): e2206053119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939700

RESUMO

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editasewt) and either Mecp2-targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2-targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2-targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.


Assuntos
Transtornos Respiratórios , Síndrome de Rett , Animais , Tronco Encefálico/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Mutação , RNA/metabolismo , Edição de RNA/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/terapia
9.
Cell Rep ; 40(5): 111156, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35926466

RESUMO

N6-methyladenosine (m6A), the most common form of RNA modification, controls CD4+ T cell homeostasis by targeting the IL-7/STAT5/SOCS signaling pathways. The role of m6A modification in unconventional T cell development remains unknown. Using mice with T cell-specific deletion of RNA methyltransferase METTL14 (T-Mettl14-/-), we demonstrate that m6A modification is indispensable for iNKT cell homeostasis. Loss of METTL14-dependent m6A modification leads to the upregulation of apoptosis in double-positive thymocytes, which in turn decreases Vα14-Jα18 gene rearrangements, resulting in drastic reduction of iNKT numbers in the thymus and periphery. Residual T-Mettl14-/- iNKT cells exhibit increased apoptosis, impaired maturation, and decreased responsiveness to IL-2/IL-15 and TCR stimulation. Furthermore, METTL14 knockdown in mature iNKT cells diminishes their cytokine production, correlating with increased Cish expression and decreased TCR signaling. Collectively, our study highlights a critical role for METTL14-dependent-m6A modification in iNKT cell development and function.


Assuntos
Células T Matadoras Naturais , Animais , Diferenciação Celular/genética , Metiltransferases , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , RNA/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
10.
Nat Commun ; 13(1): 4522, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927243

RESUMO

Genomic methods have been valuable for identifying RNA-binding proteins (RBPs) and the genes, pathways, and processes they regulate. Nevertheless, standard motif descriptions cannot be used to predict all RNA targets or test quantitative models for cellular interactions and regulation. We present a complete thermodynamic model for RNA binding to the S. cerevisiae Pumilio protein PUF4 derived from direct binding data for 6180 RNAs measured using the RNA on a massively parallel array (RNA-MaP) platform. The PUF4 model is highly similar to that of the related RBPs, human PUM2 and PUM1, with one marked exception: a single favorable site of base flipping for PUF4, such that PUF4 preferentially binds to a non-contiguous series of residues. These results are foundational for developing and testing cellular models of RNA-RBP interactions and function, for engineering RBPs, for understanding the biophysical nature of RBP binding and the evolutionary landscape of RNAs and RBPs.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Humanos , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica
11.
Methods Mol Biol ; 2516: 305-316, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35922633

RESUMO

Toeprint assays are primer extension inhibition assays that can detect the 3' end of an RNA secondary structure, the position of a bound RNA binding protein, as well as the position of a bound 30S ribosomal subunit or a stalled ribosome. Here we describe how this assay was used to identify an RNA hairpin that sequesters a Shine-Dalgarno sequence, how the RNA-binding protein CsrA can alter RNA structure and affect 30S ribosomal subunit binding, and how the macrolide antibiotic tylosin can induce ribosome stalling.


Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Ribossomos/metabolismo
12.
Cell Rep ; 40(1): 111035, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35793628

RESUMO

During the early phase of primary humoral responses, activated B cells can differentiate into different types of effector cells, dependent on B cell receptor affinity for antigen. However, the pivotal transcription factors governing these processes remain to be elucidated. Here, we show that transcription factor Bach2 protein in activated B cells is transiently induced by affinity-related signals and mechanistic target of rapamycin complex 1 (mTORC1)-dependent translation to restrain their expansion and differentiation into plasma cells while promoting memory and germinal center (GC) B cell fates. Affinity-related signals also downregulate Bach2 mRNA expression in activated B cells and their descendant memory B cells. Sustained and higher concentrations of Bach2 antagonize the GC fate. Repression of Bach2 in memory B cells predisposes their cell-fate choices upon memory recall. Our study reveals that differential dynamics of Bach2 protein and transcripts in activated B cells control their cell-fate outcomes and imprint the fates of their descendant effector cells.


Assuntos
Linfócitos B , Fatores de Transcrição de Zíper de Leucina Básica , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/genética , Centro Germinativo , RNA/metabolismo , Fatores de Transcrição/metabolismo
13.
Cell Mol Biol (Noisy-le-grand) ; 67(4): 334-339, 2022 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-35809271

RESUMO

Endometriosis is a gynecological disease that endometrial cells develop outside the uterus. This event happens when the endometrial glands grow outside the endometrium and uterine muscles, especially in the pelvis. Although endometriosis is widespread, the clinical manifestations of the disease are very different, and it is challenging to adapt to the conventional classification system to divide patients into homogeneous groups. Given the importance of endometriosis, a correct, accurate, and timely diagnosis of this disease can significantly prevent its complications. Using health-related software is one of these ways. Enhanced Endometriosis Archiving Software (ENEAS) is a web-based application based on one of the most widely used open-source database management systems (MySQL), allowing the direct link to other open-source software for data management and storage. In the current study, the effect of ENEAS application was considered in patients with endometriosis, and its influence on IL-8 and MCP-1 gene expression was evaluated. For this purpose, 100 women with endometriosis were divided into two groups of 50 patients. The first group (control group) was examined by a gynecologist and received medication and treatment. In the case group, their demographic and clinical information were entered into ENEAS software. To study the expression of the IL-8 gene and MCP-1 gene, after collecting 5 ml of blood samples in tubes containing anticoagulant, RNA extraction was performed by Total RNA Purification Kit (Cat. 17200, 37500, 17250). Then cDNA synthesis was performed for this purpose, and a Bioneer DNA synthesis kit (South Korea) was used. The results showed that the expression level of the IL-8 gene in the case group was significantly reduced compared to the control group (P = 0.035). MCP-1 gene expression was also decreased compared to the control group, but this decrease was not significant. Therefore, those who used this application for treatment had reduced expression of IL-8 and MCP-1 genes. This event indicates that this application has reduced the amount of inflammation caused by endometriosis with proper analysis.


Assuntos
Endometriose , Endometriose/tratamento farmacológico , Endometriose/genética , Endométrio/metabolismo , Feminino , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , RNA/metabolismo , Software
14.
Cell Rep ; 40(2): 111069, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35830789

RESUMO

tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs with distinct cellular functions. Here, we studied the contribution of tRFs to the regulation of postnatal ß cell maturation, a critical process that may lead to diabetes susceptibility in adulthood. We identified three tRFs abundant in neonatal rat islets originating from 5' halves (tiRNA-5s) of histidine and glutamate tRNAs. Their inhibition in these islets reduced ß cell proliferation and insulin secretion. Mitochondrial respiration was also perturbed, fitting with the mitochondrial enrichment of nuclear-encoded tiRNA-5HisGTG and tiRNA-5GluCTC. Notably, tiRNA-5 inhibition reduced Mpc1, a mitochondrial pyruvate carrier whose knock down largely phenocopied tiRNA-5 inhibition. tiRNA-5HisGTG interactome revealed binding to Musashi-1, which was essential for the mitochondrial enrichment of tiRNA-5HisGTG. Finally, tiRNA-5s were dysregulated in the islets of diabetic and diabetes-prone animals. Altogether, tiRNA-5s represent a class of regulators of ß cell maturation, and their deregulation in neonatal islets may lead to diabetes susceptibility in adulthood.


Assuntos
Células Secretoras de Insulina , RNA de Transferência , Animais , Proliferação de Células , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , RNA/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ratos
15.
Cell Rep ; 40(2): 111067, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35830799

RESUMO

The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.


Assuntos
DNA Topoisomerases Tipo I , Estruturas R-Loop , Camptotecina/farmacologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , RNA/metabolismo
16.
Front Cell Infect Microbiol ; 12: 855092, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774399

RESUMO

HIV-1 infection remains non-curative due to the latent reservoir, primarily a small pool of resting memory CD4+ T cells bearing replication-competent provirus. Pharmacological reversal of HIV-1 latency followed by intrinsic or extrinsic cell killing has been proposed as a promising strategy to target and eliminate HIV-1 viral reservoirs. Latency reversing agents have been extensively studied for their role in reactivating HIV-1 transcription in vivo, although no permanent reduction of the viral reservoir has been observed thus far. This is partly due to the complex nature of latency, which involves strict intrinsic regulation at multiple levels at transcription and RNA processing. Still, the molecular mechanisms that control HIV-1 latency establishment and maintenance have been almost exclusively studied in the context of chromatin remodeling, transcription initiation and elongation and most known LRAs target LTR-driven transcription by manipulating these. RNA metabolism is a largely understudies but critical mechanistic step in HIV-1 gene expression and latency. In this review we provide an update on current knowledge on the role of RNA processing mechanisms in viral gene expression and latency and speculate on the possible manipulation of these pathways as a therapeutic target for future cure studies.


Assuntos
Infecções por HIV , HIV-1 , Hibernação , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , RNA/metabolismo , RNA Viral/genética , Ativação Viral , Latência Viral/genética
17.
Toxins (Basel) ; 14(7)2022 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-35878208

RESUMO

Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein translation and cell death. We postulated that this depurination event could be detected using Oxford Nanopore Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin loop. In this study, A549 cells were exposed to ricin for 2-24 h in order to induce depurination. In addition, a novel software tool was developed termed RIPpore that could quantify the adenine modification of ribosomal RNA induced by ricin upon respiratory epithelial cells. We provided demonstrable evidence for the first time that this base change detected is specific to RIP activity using a neutralising antibody against ricin. We believe this represents the first detection of depurination in RNA achieved using ONT sequencers. Collectively, this work highlights the potential for ONT and direct RNA sequencing to detect and quantify depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore could have utility in the evaluation of new treatments and/or in the diagnosis of exposure to ricin.


Assuntos
Nanoporos , Ricina , Adenina/metabolismo , RNA/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo , Ricina/toxicidade , Análise de Sequência de RNA
18.
Cell Rep ; 40(3): 111115, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858552

RESUMO

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B , Animais , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , RNA/metabolismo
19.
Yi Chuan ; 44(7): 567-580, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35858769

RESUMO

DNA methylation is a type of stable epigenetic modifications that plays crucial roles in regulating gene expression, silencing transposons and maintaining genome stability. In plants, the de novo DNA methylation is established via a pathway termed as RNA-directed DNA methylation (RdDM). The plant-specific DNA-dependent RNA polymerase IV (Pol IV) as the core protein in RdDM pathway produces non-coding RNAs that direct the establishment of DNA methylation, regulates gene expression and controls plant development. Pol IV function is regulated by several proteins including SHH1, which recognizes H3K9 methylation and guides Pol IV to genome specific sites, the chromatin remodeling factor CLSY family that is involved in assisting Pol IV chromatin association and RDR2 that converts Pol IV produced single-stranded RNA into double-stranded RNA. In this review, we summarize the latest progress on Pol IV and its co-regulators, and focus on their functions in shaping epigenome and development in plants, which might provide implications for studying of DNA methylation and crop breeding.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilação de DNA , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Plantas/genética , Plantas/metabolismo , RNA/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética
20.
Life Sci Alliance ; 5(11)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35777956

RESUMO

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Esclerose Amiotrófica Lateral , Proteínas Relacionadas à Autofagia , Demência Frontotemporal , Proteínas Associadas aos Microtúbulos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteômica , RNA/genética , RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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