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1.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205049

RESUMO

Aberrant RNA-protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA-protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.


Assuntos
Peptídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Processos Fotoquímicos , Ligação Proteica/efeitos dos fármacos , RNA/química , Proteínas de Ligação a RNA/química
2.
Nat Commun ; 12(1): 4231, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244499

RESUMO

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.


Assuntos
Amiloide/metabolismo , Agregação Patológica de Proteínas/patologia , RNA/metabolismo , Proteínas tau/metabolismo , Amiloide/ultraestrutura , Técnicas Biossensoriais , Humanos , Ressonância Magnética Nuclear Biomolecular , RNA/ultraestrutura , RNA Fúngico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/isolamento & purificação , Proteínas tau/ultraestrutura
3.
Bioinformatics ; 37(Suppl_1): i222-i230, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252943

RESUMO

MOTIVATION: Increasing evidence suggests that post-transcriptional ribonucleic acid (RNA) modifications regulate essential biomolecular functions and are related to the pathogenesis of various diseases. Precise identification of RNA modification sites is essential for understanding the regulatory mechanisms of RNAs. To date, many computational approaches for predicting RNA modifications have been developed, most of which were based on strong supervision enabled by base-resolution epitranscriptome data. However, high-resolution data may not be available. RESULTS: We propose WeakRM, the first weakly supervised learning framework for predicting RNA modifications from low-resolution epitranscriptome datasets, such as those generated from acRIP-seq and hMeRIP-seq. Evaluations on three independent datasets (corresponding to three different RNA modification types and their respective sequencing technologies) demonstrated the effectiveness of our approach in predicting RNA modifications from low-resolution data. WeakRM outperformed state-of-the-art multi-instance learning methods for genomic sequences, such as WSCNN, which was originally designed for transcription factor binding site prediction. Additionally, our approach captured motifs that are consistent with existing knowledge, and visualization of the predicted modification-containing regions unveiled the potentials of detecting RNA modifications with improved resolution. AVAILABILITY IMPLEMENTATION: The source code for the WeakRM algorithm, along with the datasets used, are freely accessible at: https://github.com/daiyun02211/WeakRM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
RNA , Software , Algoritmos , Ligação Proteica , RNA/genética , RNA/metabolismo , Análise de Sequência de RNA , Aprendizado de Máquina Supervisionado
4.
Bioinformatics ; 37(Suppl_1): i308-i316, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252974

RESUMO

MOTIVATION: Understanding how proteins recognize their RNA targets is essential to elucidate regulatory processes in the cell. Many RNA-binding proteins (RBPs) form complexes or have multiple domains that allow them to bind to RNA in a multivalent, cooperative manner. They can thereby achieve higher specificity and affinity than proteins with a single RNA-binding domain. However, current approaches to de novo discovery of RNA binding motifs do not take multivalent binding into account. RESULTS: We present Bipartite Motif Finder (BMF), which is based on a thermodynamic model of RBPs with two cooperatively binding RNA-binding domains. We show that bivalent binding is a common strategy among RBPs, yielding higher affinity and sequence specificity. We furthermore illustrate that the spatial geometry between the binding sites can be learned from bound RNA sequences. These discovered bipartite motifs are consistent with previously known motifs and binding behaviors. Our results demonstrate the importance of multivalent binding for RNA-binding proteins and highlight the value of bipartite motif models in representing the multivalency of protein-RNA interactions. AVAILABILITY AND IMPLEMENTATION: BMF source code is available at https://github.com/soedinglab/bipartite_motif_finder under a GPL license. The BMF web server is accessible at https://bmf.soedinglab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Proteínas de Ligação a RNA , Software , Sítios de Ligação , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Termodinâmica
5.
J Med Chem ; 64(11): 7110-7155, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34060847

RESUMO

RNAs are involved in an enormous range of cellular processes, including gene regulation, protein synthesis, and cell differentiation, and dysfunctional RNAs are associated with disorders such as cancers, neurodegenerative diseases, and viral infections. Thus, the identification of compounds with the ability to bind RNAs and modulate their functions is an exciting approach for developing next-generation therapies. Numerous RNA-binding agents have been reported over the past decade, but the design of synthetic molecules with selectivity for specific RNA sequences is still in its infancy. In this perspective, we highlight recent advances in targeting RNAs with synthetic molecules, and we discuss the potential value of this approach for the development of innovative therapeutic agents.


Assuntos
Descoberta de Drogas , RNA/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , RNA/antagonistas & inibidores , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
6.
Mol Cell ; 81(11): 2275-2277, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34087179

RESUMO

Wan et al. (2021) establish a powerful new platform to measure the dynamics of transcription and splicing of endogenous genes in single cells in real time. Combining real-time measurements with multiple deep-sequencing tools reveals an unexpectedly high amount of spliceosome activity, prompting a reconsideration of current models of how introns are removed from pre-mRNA.


Assuntos
RNA , Spliceossomos , Íntrons/genética , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/genética , Spliceossomos/metabolismo
7.
Nat Commun ; 12(1): 3368, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099723

RESUMO

Folding of RNA can produce elaborate tertiary structures, corresponding to their diverse roles in the regulation of biological activities. Direct observation of RNA structures at high resolution in their native form however remains a challenge. The large vestibule and the narrow constriction of a Mycobacterium smegmatis porin A (MspA) suggests a sensing mode called nanopore trapping/translocation, which clearly distinguishes between microRNA, small interfering RNA (siRNA), transfer RNA (tRNA) and 5 S ribosomal RNA (rRNA). To further profit from the acquired event characteristics, a custom machine learning algorithm is developed. Events from measurements with a mixture of RNA analytes can be automatically classified, reporting a general accuracy of ~93.4%. tRNAs, which possess a unique tertiary structure, report a highly distinguishable sensing feature, different from all other RNA types tested in this study. With this strategy, tRNAs from different sources are measured and a high structural conservation across different species is observed in single molecule.


Assuntos
Mycobacterium smegmatis/metabolismo , Nanoporos , Porinas/metabolismo , RNA/metabolismo , Aprendizado de Máquina , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Peso Molecular , Mycobacterium smegmatis/genética , Conformação de Ácido Nucleico , Porinas/química , Porinas/genética , RNA/química , RNA/genética , Dobramento de RNA , Transporte de RNA , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo
8.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34110129

RESUMO

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Ebolavirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Alanina/genética , Alanina/metabolismo , Antivirais/metabolismo , Pareamento de Bases , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
9.
Nat Commun ; 12(1): 3370, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099647

RESUMO

A sensitive approach to quantitative analysis of transcriptional regulation in diploid organisms is analysis of allelic imbalance (AI) in RNA sequencing (RNA-seq) data. A near-universal practice in such studies is to prepare and sequence only one library per RNA sample. We present theoretical and experimental evidence that data from a single RNA-seq library is insufficient for reliable quantification of the contribution of technical noise to the observed AI signal; consequently, reliance on one-replicate experimental design can lead to unaccounted-for variation in error rates in allele-specific analysis. We develop a computational approach, Qllelic, that accurately accounts for technical noise by making use of replicate RNA-seq libraries. Testing on new and existing datasets shows that application of Qllelic greatly decreases false positive rate in allele-specific analysis while conserving appropriate signal, and thus greatly improves reproducibility of AI estimates. We explore sources of technical overdispersion in observed AI signal and conclude by discussing design of RNA-seq studies addressing two biologically important questions: quantification of transcriptome-wide AI in one sample, and differential analysis of allele-specific expression between samples.


Assuntos
Desequilíbrio Alélico , Biblioteca Gênica , Polimorfismo de Nucleotídeo Único , RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Algoritmos , Alelos , Animais , Feminino , Camundongos da Linhagem 129 , Modelos Genéticos , RNA/metabolismo
10.
Nucleic Acids Res ; 49(W1): W72-W79, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34086933

RESUMO

Prediction of protein-RNA interactions is important to understand post-transcriptional events taking place in the cell. Here we introduce catRAPID omics v2.0, an update of our web server dedicated to the computation of protein-RNA interaction propensities at the transcriptome- and RNA-binding proteome-level in 8 model organisms. The server accepts multiple input protein or RNA sequences and computes their catRAPID interaction scores on updated precompiled libraries. Additionally, it is now possible to predict the interactions between a custom protein set and a custom RNA set. Considerable effort has been put into the generation of a new database of RNA-binding motifs that are searched within the predicted RNA targets of proteins. In this update, the sequence fragmentation scheme of the catRAPID fragment module has been included, which allows the server to handle long linear RNAs and to analyse circular RNAs. For the top-scoring protein-RNA pairs, the web server shows the predicted binding sites in both protein and RNA sequences and reports whether the predicted interactions are conserved in orthologous protein-RNA pairs. The catRAPID omics v2.0 web server is a powerful tool for the characterization and classification of RNA-protein interactions and is freely available at http://service.tartaglialab.com/page/catrapid_omics2_group along with documentation and tutorial.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Software , Animais , Sítios de Ligação , Humanos , Camundongos , RNA/química , RNA Circular/química , RNA Circular/metabolismo , Proteínas de Ligação a RNA/química , Ratos , Análise de Sequência de Proteína , Análise de Sequência de RNA
11.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
12.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
13.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065193

RESUMO

The scientific interest in the beneficial properties of natural substances has been recognized for decades, as well as the growing attention in extracellular vesicles (EVs) released by different organisms, in particular from animal cells. However, there is increasing interest in the isolation and biological and functional characterization of these lipoproteic structures in the plant kingdom. Similar to animal vesicles, these plant-derived extracellular vesicles (PDEVs) exhibit a complex content of small RNAs, proteins, lipids, and other metabolites. This sophisticated composition enables PDEVs to be therapeutically attractive. In this review, we report and discuss current knowledge on PDEVs in terms of isolation, characterization of their content, biological properties, and potential use as drug delivery systems. In conclusion, we outline controversial issues on which the scientific community shall focus the attention shortly.


Assuntos
Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Plantas/metabolismo , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipídeos/fisiologia , Proteínas/metabolismo , RNA/metabolismo
14.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
15.
BMC Bioinformatics ; 22(1): 288, 2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34051729

RESUMO

BACKGROUND: As a common and abundant RNA methylation modification, N6-methyladenosine (m6A) is widely spread in various species' transcriptomes, and it is closely related to the occurrence and development of various life processes and diseases. Thus, accurate identification of m6A methylation sites has become a hot topic. Most biological methods rely on high-throughput sequencing technology, which places great demands on the sequencing library preparation and data analysis. Thus, various machine learning methods have been proposed to extract various types of features based on sequences, then occupied conventional classifiers, such as SVM, RF, etc., for m6A methylation site identification. However, the identification performance relies heavily on the extracted features, which still need to be improved. RESULTS: This paper mainly studies feature extraction and classification of m6A methylation sites in a natural language processing way, which manages to organically integrate the feature extraction and classification simultaneously, with consideration of upstream and downstream information of m6A sites. One-hot, RNA word embedding, and Word2vec are adopted to depict sites from the perspectives of the base as well as its upstream and downstream sequence. The BiLSTM model, a well-known sequence model, was then constructed to discriminate the sequences with potential m6A sites. Since the above-mentioned three feature extraction methods focus on different perspectives of m6A sites, an ensemble deep learning predictor (EDLm6APred) was finally constructed for m6A site prediction. Experimental results on human and mouse data sets show that EDLm6APred outperforms the other single ones, indicating that base, upstream, and downstream information are all essential for m6A site detection. Compared with the existing m6A methylation site prediction models without genomic features, EDLm6APred obtains 86.6% of the area under receiver operating curve on the human data sets, indicating the effectiveness of sequential modeling on RNA. To maximize user convenience, a webserver was developed as an implementation of EDLm6APred and made publicly available at www.xjtlu.edu.cn/biologicalsciences/EDLm6APred . CONCLUSIONS: Our proposed EDLm6APred method is a reliable predictor for m6A methylation sites.


Assuntos
Aprendizado Profundo , Adenosina/metabolismo , Animais , Metilação , Camundongos , RNA/metabolismo , RNA Mensageiro
16.
Nat Commun ; 12(1): 2639, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976128

RESUMO

The placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application ( https://www.obgyn.cam.ac.uk/placentome/ ).


Assuntos
Retardo do Crescimento Fetal/genética , Placenta/patologia , Pré-Eclâmpsia/genética , RNA/genética , Transcriptoma/genética , Biópsia , Conjuntos de Dados como Assunto , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/patologia , Proteínas Relacionadas à Folistatina/sangue , Proteínas Relacionadas à Folistatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Placenta/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/patologia , Gravidez , RNA/metabolismo , RNA-Seq
17.
Methods Mol Biol ; 2290: 229-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009594

RESUMO

Photosynthetic cyanobacteria are not only model organisms for studying photosynthesis and biological cycling of carbon in biosphere but also potential "green microbial factories" to produce renewable fuels and chemicals, due to their capability to utilizing solar energy and CO2. Therefore, strategies for gene regulation and carbon flux redirection are important for both fundamental research and metabolic engineering of cyanobacteria. To address the challenges, regulatory tools based on artificial small RNAs have been developed with satisfactory effects for single or multiple gene(s) regulation in various cyanobacterial species. When combined with the promoters of varying gradient strength and the inducible switches developed in recent years, it is now feasible to realize precise gene regulation in photosynthetic cyanobacteria for producing fuels and chemicals. Here in this chapter, we provide a detailed introduction of the design principles and constructing methods of the artificial sRNA tools to achieve accurate inducible regulation of cyanobacterial gene(s).


Assuntos
Cianobactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Pequeno RNA não Traduzido/síntese química , Biocombustíveis , Carbono/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , Expressão Gênica/genética , Engenharia Metabólica/métodos , Fotossíntese/fisiologia , RNA/metabolismo , Pequeno RNA não Traduzido/metabolismo , Biologia Sintética/métodos
18.
Nat Methods ; 18(5): 507-519, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33963355

RESUMO

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Análise de Célula Única/métodos , Animais , Sítios de Ligação , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Sequenciamento por Nanoporos , RNA/química , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA , Transcriptoma
19.
Nucleic Acids Res ; 49(W1): W67-W71, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34038531

RESUMO

The interaction between RNA and RNA-binding proteins (RBPs) has a key role in the regulation of gene expression, in RNA stability, and in many other biological processes. RBPs accomplish these functions by binding target RNA molecules through specific sequence and structure motifs. The identification of these binding motifs is therefore fundamental to improve our knowledge of the cellular processes and how they are regulated. Here, we present BRIO (BEAM RNA Interaction mOtifs), a new web server designed for the identification of sequence and structure RNA-binding motifs in one or more RNA molecules of interest. BRIO enables the user to scan over 2508 sequence motifs and 2296 secondary structure motifs identified in Homo sapiens and Mus musculus, in three different types of experiments (PAR-CLIP, eCLIP, HITS). The motifs are associated with the binding of 186 RBPs and 69 protein domains. The web server is freely available at http://brio.bio.uniroma2.it.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/química , Software , Animais , Sequência de Bases , Linhagem Celular , Humanos , Internet , Camundongos , Motivos de Nucleotídeos , RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Viral/metabolismo , Análise de Sequência de RNA
20.
Aging (Albany NY) ; 13(8): 11919-11941, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33952721

RESUMO

M6A-related genes have been proven to play an important role in many cancers. However, the role of that in adrenocortical carcinoma (ACC) has not been fully elucidated. In the present study, 77 ACC samples from TCGA database were divided into localized (n = 46) and metastatic (n = 31) groups. Three differential expression genes (DEGs) and five prognostic m6A genes were screened out. M6A-related risk signature (RBM15 and HNRNPC) was constructed by the Lasso regression analysis. In TCGA cohort (training cohort), the risk signature was identified as an ACC-independent prognostic factor and can distinguish the prognostic difference of ACC patients with clinical stage I-II, T3-4 and N0 stages. A nomogram combining T stage and m6A risk score was constructed to predict the overall survival rate (OSR) of individual at 1,2,3 year. Meanwhile, its prognostic value was also confirmed in the validation cohort (GSE33371 dataset). The potential associations between m6A risk level and immune checkpoint inhibitors (ICIs) therapy were also investigated via the TISIDB online tool. High m6A risk not only can suppress immunotherapy-related biological processes, but also repress the expressions of immune-checkpoint markers. Moreover, five pairs of clinical specimens were collected to confirm the overexpression of HNRNPC and non-ectopic expression of RBM15 in tumor tissues. HNRNPC was proven to promote the proliferation, migration and invasion of H295R and SW13 cells through MTT and Transwell assays. In conclusion, the m6A-related risk signature was beneficial for prognostic analysis and can affect immune microenvironment in ACC. HNRNPC played a pro-cancer role in ACC progression.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Biomarcadores Tumorais/genética , Epigênese Genética , Nomogramas , Adenosina/análogos & derivados , Adenosina/metabolismo , Córtex Suprarrenal/imunologia , Córtex Suprarrenal/patologia , Córtex Suprarrenal/cirurgia , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/mortalidade , Neoplasias do Córtex Suprarrenal/terapia , Adrenalectomia , Carcinoma Adrenocortical/diagnóstico , Carcinoma Adrenocortical/mortalidade , Carcinoma Adrenocortical/terapia , Idoso , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Biologia Computacional , Conjuntos de Dados como Assunto , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Masculino , Metilação , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Taxa de Sobrevida , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
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