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1.
Biol Lett ; 20(7): 20240147, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38982851

RESUMO

The nucleus interacts with the other organelles to perform essential functions of the eukaryotic cell. Mitochondria have their own genome and communicate back to the nucleus in what is known as mitochondrial retrograde response. Information is transferred to the nucleus in many ways, leading to wide-ranging changes in nuclear gene expression and culminating with changes in metabolic, regulatory or stress-related pathways. RNAs are emerging molecules involved in this signalling. RNAs encode precise information and are involved in highly target-specific signalling, through a wide range of processes known as RNA interference. RNA-mediated mitochondrial retrograde response requires these molecules to exit the mitochondrion, a process that is still mostly unknown. We suggest that the proteins/complexes translocases of the inner membrane, polynucleotide phosphorylase, mitochondrial permeability transition pore, and the subunits of oxidative phosphorylation complexes may be responsible for RNA export.


Assuntos
Núcleo Celular , Mitocôndrias , Mitocôndrias/metabolismo , Núcleo Celular/metabolismo , RNA/metabolismo , RNA/genética , Animais , Transporte de RNA , Células Eucarióticas/metabolismo , Eucariotos/metabolismo , Eucariotos/genética , Eucariotos/fisiologia , Transdução de Sinais
2.
Commun Biol ; 7(1): 823, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38971915

RESUMO

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated and experimental datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and in-situ cell type calling. ComSeg is available as a documented and open source pip package at https://github.com/fish-quant/ComSeg .


Assuntos
Algoritmos , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Processamento de Imagem Assistida por Computador/métodos , Humanos , Animais , Software , RNA/genética , Hibridização in Situ Fluorescente/métodos
3.
Sci Rep ; 14(1): 16018, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992080

RESUMO

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.


Assuntos
Glicina , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Glicina/metabolismo , Glicina/química , RNA/metabolismo , RNA/genética , Domínios Proteicos , Mutação , Sítios de Ligação , Humanos , Separação de Fases , Proteínas de Arabidopsis
4.
Theranostics ; 14(10): 3827-3842, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38994027

RESUMO

Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.


Assuntos
Sistemas CRISPR-Cas , Dependovirus , Técnicas de Silenciamento de Genes , Espermatogênese , Testículo , Masculino , Animais , Dependovirus/genética , Sistemas CRISPR-Cas/genética , Camundongos , Testículo/metabolismo , Técnicas de Silenciamento de Genes/métodos , Espermatogênese/genética , RNA/genética , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem
5.
Med Sci (Paris) ; 40(6-7): 525-533, 2024.
Artigo em Francês | MEDLINE | ID: mdl-38986097

RESUMO

Many diseases originate from either the absence or defective expression of a given protein. For some of them, the lacking protein is secreted or can be taken up by cells when delivered exogenously. In such cases, therapies initially involved administering the physiological protein extracted from human tissues. Subsequently, genetic engineering enabled the production of proteins through cell fermentation after introducing the corresponding gene. For many other pathologies, the deficient protein cannot be delivered exogenously. Thus, an endogenous production of the therapeutic protein by the cells themselves is necessary. Messenger RNA (mRNA) technology, like its predecessor DNA, aims to supplement the genetic information needed to produce the therapeutic protein within the cells. However, unlike DNA-based therapies, mRNA transfer allows for transient expression of the protein of interest, which offers an advantage in numerous pathologies. Nonetheless, mastering the quantity, quality, and spatio-temporal regulation of protein production encoded by therapeutic mRNA remains a significant challenge for the development of this approach.


Title: « ReNAissance ¼1 des biothérapies par ARN. Abstract: Nombre de maladies ont pour origine une absence d'expression ou une expression défectueuse d'une protéine donnée. Pour certaines d'entre elles, la protéine faisant défaut est circulante et peut être captée par les cellules lorsqu'elle est délivrée de façon exogène. Dans ce cas, les thérapies ont d'abord consisté en l'administration de la protéine thérapeutique extraite de tissus humains. Par la suite, le génie génétique a permis la production des protéines par fermentation de cellules après y avoir introduit le gène correspondant. Pour beaucoup d'autres maladies, la protéine faisant défaut ne peut être délivrée de façon exogène. Une production endogène de la protéine thérapeutique, par les cellules elles-mêmes est donc nécessaire. La technologie de l'ARN messager (ARNm), comme celle la précédant de l'ADN, se propose de supplémenter, au cœur des cellules, l'information génétique nécessaire pour produire elles-mêmes la protéine thérapeutique. Cependant, contrairement aux thérapies utilisant l'ADN, le transfert d'ARNm permet une expression transitoire de la protéine d'intérêt ce qui constitue un avantage dans nombre de maladies. La maîtrise de la quantité, de la qualité et de la régulation spatio-temporelle de la production d'une protéine codée par l'ARNm thérapeutique représente, néanmoins, un défi important pour le développement de cette approche.


Assuntos
RNA Mensageiro , Humanos , RNA Mensageiro/genética , Terapia Genética/métodos , Terapia Genética/tendências , Animais , Terapia Biológica/métodos , Terapia Biológica/tendências , RNA/genética
6.
Endocrinology ; 165(8)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38946397

RESUMO

Uterine leiomyoma or fibroids are prevalent noncancerous tumors of the uterine muscle layer, yet their origin and development remain poorly understood. We analyzed RNA expression profiles of 15 epigenetic mediators in uterine fibroids compared to myometrium using publicly available RNA sequencing (RNA-seq) data. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key N6-methyladenosine (m6A) modifiers in fibroids and their matched myometrium, showing no significant differences in concordance with our RNA expression profiles. To determine RNA modification abundance, mRNA and small RNA from fibroids and matched myometrium were analyzed by ultra-high performance liquid chromatography-mass spectrometry identifying prevalent m6A and 11 other known modifiers. However, no aberrant expression in fibroids was detected. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic subtype. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression, and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and diverse patient cohort.


Assuntos
Adenosina , Leiomioma , Neoplasias Uterinas , Leiomioma/genética , Leiomioma/metabolismo , Humanos , Feminino , Adenosina/análogos & derivados , Adenosina/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Miométrio/metabolismo , Miométrio/patologia , Pessoa de Meia-Idade , Adulto , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Epigênese Genética
7.
Sci Transl Med ; 16(754): eadl3848, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38959329

RESUMO

Autoantibodies to nuclear antigens are hallmarks of systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to this autoimmune disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second-most prevalent isotype in serum and, along with IgG, is deposited in glomeruli in individuals with lupus nephritis. We show that individuals with SLE have serum IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoprotein (Sm/RNP), played a role in IC activation of pDCs. We found that pDCs expressed the IgA-specific Fc receptor, FcαR, and IgA1 autoantibodies synergized with IgG in RNA-containing ICs to generate robust primary blood pDC IFN-α responses in vitro. pDC responses to these ICs required both FcαR and FcγRIIa, showing synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. Circulating pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Although pDC FcαR expression correlated with the blood IFN-stimulated gene signature in SLE, Toll-like receptor 7 agonists, but not IFN-α, up-regulated pDC FcαR expression in vitro. Together, we show a mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.


Assuntos
Complexo Antígeno-Anticorpo , Autoanticorpos , Células Dendríticas , Imunoglobulina A , Imunoglobulina G , Lúpus Eritematoso Sistêmico , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina A/sangue , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/sangue , RNA/metabolismo , Feminino , Interferon-alfa/metabolismo , Adulto , Receptores Fc/metabolismo , Receptores Fc/imunologia , Receptor 7 Toll-Like/metabolismo , Masculino , Receptores de IgG/metabolismo
8.
Sci Rep ; 14(1): 16859, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39039071

RESUMO

Confirmatory diagnosis of childhood tuberculosis (TB) remains a challenge mainly due to its dependence on sputum samples and the paucibacillary nature of the disease. Thus, only ~ 30% of suspected cases in children are diagnosed and the need for minimally invasive, non-sputum-based biomarkers remains unmet. Understanding host molecular changes by measuring blood-based transcriptomic markers has shown promise as a diagnostic tool for TB. However, the implication of sex contributing to disease heterogeneity and therefore diagnosis remains to be understood. Using publicly available gene expression data (GSE39939, GSE39940; n = 370), we report a sex-specific RNA biomarker signature that could improve the diagnosis of TB disease in children. We found four gene biomarker signatures for male (SLAMF8, GBP2, WARS, and FCGR1C) and female pediatric patients (GBP6, CELSR3, ALDH1A1, and GBP4) from Kenya, South Africa, and Malawi. Both signatures achieved a sensitivity of 85% and a specificity of 70%, which approaches the WHO-recommended target product profile for a triage test. Our gene signatures outperform most other gene signatures reported previously for childhood TB diagnosis.


Assuntos
Biomarcadores , Tuberculose , Humanos , Feminino , Masculino , Criança , Biomarcadores/sangue , Tuberculose/diagnóstico , Tuberculose/genética , Tuberculose/sangue , RNA/genética , Pré-Escolar , Transcriptoma , Fatores Sexuais , Perfilação da Expressão Gênica , Adolescente
9.
PLoS One ; 19(7): e0302772, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39042659

RESUMO

Noncoding RNAs play a part in many chronic diseases and interact with each other to regulate gene expression. MicroRNA-9-5p (miR9) has been thought to be a potential inhibitor of diabetic cardiomyopathy. Here we examined the role of miR9 in regulating cardiac fibrosis in the context of diabetic cardiomyopathy. We further expanded our studies through investigation of a regulatory circularRNA, circRNA_012164, on the action of miR9. We showed at both the in vivo and in vitro level that glucose induced downregulation of miR9 and upregulation of circRNA_012164 resulted in the subsequent upregulation of downstream fibrotic genes. Further, knockdown of circRNA_012164 shows protective effects in cardiac endothelial cells and reverses increased transcription of genes associated with fibrosis and fibroblast proliferation through a regulatory axis with miR9. This study presents a novel regulatory axis involving noncoding RNA that is evidently important in the development of cardiac fibrosis in diabetic cardiomyopathy.


Assuntos
Cardiomiopatias Diabéticas , Fibrose , MicroRNAs , RNA Circular , MicroRNAs/genética , MicroRNAs/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Animais , RNA Circular/genética , RNA Circular/metabolismo , Camundongos , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , RNA/genética , RNA/metabolismo , Glucose/metabolismo , Regulação da Expressão Gênica , Proliferação de Células/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ratos , Camundongos Endogâmicos C57BL
10.
Proc Natl Acad Sci U S A ; 121(31): e2220020121, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39042693

RESUMO

Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity.


Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Quadruplex G , Proteínas de Ligação a RNA , RNA , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Biossíntese de Proteínas , RNA/metabolismo , RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
11.
J Am Chem Soc ; 146(29): 19686-19689, 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-38991204

RESUMO

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.


Assuntos
Hibridização de Ácido Nucleico , RNA , Termodinâmica , RNA/química , Cinética , Conformação de Ácido Nucleico , Pareamento de Bases , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Separação de Fases
12.
Genome Res ; 34(6): 822-836, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39009472

RESUMO

N 6-Methyladenosine (m6A) is a prevalent and highly regulated RNA modification essential for RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m6A is implicated in neurogenesis and learning. Although extensively studied, its presence in specific cell types remains poorly understood. We investigated m6A in the hippocampus at a single-cell resolution, revealing a comprehensive landscape of m6A modifications within individual cells. Through our analysis, we uncovered transcripts exhibiting a dense m6A profile, notably linked to neurological disorders such as Alzheimer's disease. Our findings suggest a pivotal role of m6A-containing transcripts, particularly in the context of CAMK2A neurons. Overall, this work provides new insights into the molecular mechanisms underlying hippocampal physiology and lays the foundation for future studies investigating the dynamic nature of m6A RNA methylation in the healthy and diseased brain.


Assuntos
Adenosina , Hipocampo , Análise de Célula Única , Hipocampo/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Análise de Célula Única/métodos , Camundongos , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Metilação , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , RNA/metabolismo , RNA/genética , Humanos , Metilação de RNA
13.
Nat Commun ; 15(1): 5725, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977675

RESUMO

The rational targeting of RNA with small molecules is hampered by our still limited understanding of RNA structural and dynamic properties. Most in silico tools for binding site identification rely on static structures and therefore cannot face the challenges posed by the dynamic nature of RNA molecules. Here, we present SHAMAN, a computational technique to identify potential small-molecule binding sites in RNA structural ensembles. SHAMAN enables exploring the conformational landscape of RNA with atomistic molecular dynamics simulations and at the same time identifying RNA pockets in an efficient way with the aid of probes and enhanced-sampling techniques. In our benchmark composed of large, structured riboswitches as well as small, flexible viral RNAs, SHAMAN successfully identifies all the experimentally resolved pockets and ranks them among the most favorite probe hotspots. Overall, SHAMAN sets a solid foundation for future drug design efforts targeting RNA with small molecules, effectively addressing the long-standing challenges in the field.


Assuntos
Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , RNA Viral , RNA , Sítios de Ligação , RNA/química , RNA/metabolismo , RNA Viral/química , RNA Viral/metabolismo , RNA Viral/genética , Riboswitch , Bibliotecas de Moléculas Pequenas/química , Profissionais de Medicina Tradicional
14.
BMC Biol ; 22(1): 151, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977974

RESUMO

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.


Assuntos
Citidina Desaminase , Switching de Imunoglobulina , Estruturas R-Loop , Switching de Imunoglobulina/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/química , Animais , Camundongos , Metilação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Recombinação Genética , RNA/metabolismo , RNA/genética
15.
Brief Bioinform ; 25(4)2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38980372

RESUMO

Around 50 years ago, molecular biology opened the path to understand changes in forms, adaptations, complexity, or the basis of human diseases through myriads of reports on gene birth, gene duplication, gene expression regulation, and splicing regulation, among other relevant mechanisms behind gene function. Here, with the advent of big data and artificial intelligence (AI), we focus on an elusive and intriguing mechanism of gene function regulation, RNA editing, in which a single nucleotide from an RNA molecule is changed, with a remarkable impact in the increase of the complexity of the transcriptome and proteome. We present a new generation approach to assess the functional conservation of the RNA-editing targeting mechanism using two AI learning algorithms, random forest (RF) and bidirectional long short-term memory (biLSTM) neural networks with an attention layer. These algorithms, combined with RNA-editing data coming from databases and variant calling from same-individual RNA and DNA-seq experiments from different species, allowed us to predict RNA-editing events using both primary sequence and secondary structure. Then, we devised a method for assessing conservation or divergence in the molecular mechanisms of editing completely in silico: the cross-testing analysis. This novel method not only helps to understand the conservation of the editing mechanism through evolution but could set the basis for achieving a better understanding of the adenosine-targeting mechanism in other fields.


Assuntos
Aprendizado de Máquina , Edição de RNA , Humanos , Algoritmos , Simulação por Computador , Biologia Computacional/métodos , Redes Neurais de Computação , RNA/genética , RNA/metabolismo
16.
Cell Genom ; 4(7): 100603, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38955188

RESUMO

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.


Assuntos
Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Humanos , Imunoprecipitação/métodos , Sítios de Ligação , Software , Biologia Computacional/métodos , RNA/metabolismo , RNA/genética , Ligação Proteica
17.
Sci Rep ; 14(1): 15789, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38982195

RESUMO

Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.


Assuntos
COVID-19 , Eritrócitos , SARS-CoV-2 , Receptor 7 Toll-Like , Humanos , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Eritrócitos/metabolismo , COVID-19/virologia , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Sepse/metabolismo , Sepse/sangue , Sepse/genética , Membrana Eritrocítica/metabolismo , Masculino , RNA/metabolismo , RNA/genética , Feminino
18.
Database (Oxford) ; 20242024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38970503

RESUMO

The relationship between different ribonucleic acids (RNAs) and tumor immunity has been widely investigated. However, a systematic description of tumor immune-related RNAs in different tumors is still lacking. We collected the relationship of tumor immune-related RNAs from the published literature and presented them in a user-friendly interface, "ImmRNA" (http://www.immrna.cn/), to provide a resource to study immune-RNA-cancer regulatory relations. The ImmRNA contains 49 996 curated entries. Each entry includes gene symbols, gene types, target genes, downstream effects, functions, immune cells, and other information. By rearranging and reanalyzing the data, our dataset contains the following key points: (i) providing the links between RNAs and the immune in cancers, (ii) displaying the downstream effects and functions of RNAs, (iii) listing immune cells and immune pathways related to RNA function, (iv) showing the relationship between RNAs and prognostic outcomes, and (v) exhibiting the experimental methods described in the article. ImmRNA provides a valuable resource for understanding the functions of tumor immune-related RNAs. Database URL:  http://www.immrna.cn/.


Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/imunologia , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , RNA Neoplásico/genética , RNA Neoplásico/imunologia , RNA/genética , RNA/imunologia
19.
Chem Commun (Camb) ; 60(59): 7610-7613, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-38957007

RESUMO

Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.


Assuntos
Benzo(a)pireno , Corantes Fluorescentes , Mutação Puntual , RNA , Benzo(a)pireno/análise , Benzo(a)pireno/química , RNA/genética , RNA/química , RNA/análise , Corantes Fluorescentes/química , Colorimetria , Espectrometria de Fluorescência , Oligonucleotídeos/química
20.
Nat Commun ; 15(1): 5941, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39009595

RESUMO

Recent development of RNA velocity uses master equations to establish the kinetics of the life cycle of RNAs from unspliced RNA to spliced RNA (i.e., mature RNA) to degradation. To feed this kinetic analysis, simultaneous measurement of unspliced RNA and spliced RNA in single cells is greatly desired. However, the majority of single-cell RNA-seq chemistry primarily captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based single-cell RNA-seq method: snapTotal-seq. We benchmark this method with multiple single-cell RNA-seq assays in their performance in kinetic analysis of cell cycle by RNA velocity. Next, with LASSO regression between transcription factors, we identify the critical regulatory hubs mediating the cell cycle dynamics. We also apply snapTotal-seq to profile the oncogene-induced senescence and identify the key regulatory hubs governing the entry of senescence. Furthermore, from the comparative analysis of unspliced RNA and spliced RNA, we identify a significant portion of genes whose expression changes occur in spliced RNA but not to the same degree in unspliced RNA, indicating these gene expression changes are mainly controlled by post-transcriptional regulation. Overall, we demonstrate that snapTotal-seq can provide enriched information about gene regulation, especially during the transition between cell states.


Assuntos
Ciclo Celular , RNA , Análise de Célula Única , Fatores de Transcrição , Análise de Célula Única/métodos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Humanos , Ciclo Celular/genética , RNA/metabolismo , RNA/genética , Splicing de RNA , Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Senescência Celular/genética , RNA-Seq/métodos , Cinética
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