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1.
Nat Commun ; 12(1): 89, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397958

RESUMO

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.


Assuntos
Processamento Alternativo/genética , Desenvolvimento Muscular/genética , Contração Miocárdica/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinina/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
2.
J Vis Exp ; (166)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33369604

RESUMO

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.


Assuntos
Galectina 3/genética , Microesferas , Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Núcleo Celular/metabolismo , Galectina 3/metabolismo , Células HeLa , Humanos , Peptídeos/metabolismo , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética
3.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2357-2360, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018480

RESUMO

In the past decades an extensive mathematical literature was developed to model and analyze gene networks under both deterministic and stochastic formalisms. However, such literature is predominantly focused to deal with the modeling of transcriptional and translational regulation, but results related to post-transcriptional regulation and its connection with transcriptional regulation are poorly investigated. However, it is becoming of paramount importance the need for modeling post-transcriptional regulation via splicing especially for minor organisms or viruses.The aim of this study is to propose a first general basic modeling scheme for modeling gene expression via alternative splicing and investigating the basic deterministic and stochastic features of the pre-mRNA, mRNAs and proteins under different biological conditions.This first study showed the dynamical properties of alternative splicing, the faster kinetics of the pre-mRNA compared to the mRNA and the importance to stochastically model gene networks when considering the post-transcriptional regulation.


Assuntos
Precursores de RNA , Processamento de RNA , Processamento Alternativo , Redes Reguladoras de Genes , Precursores de RNA/metabolismo , Processamento de RNA/genética , RNA Mensageiro/genética
4.
Nat Commun ; 11(1): 4455, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901005

RESUMO

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Mitose/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética
5.
Mol Cell ; 80(2): 227-236.e5, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32991829

RESUMO

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Precursores de RNA/metabolismo , Ribonucleases/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Precursores de RNA/ultraestrutura , Ribonucleases/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Especificidade por Substrato
6.
Gene ; 760: 145021, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32763489

RESUMO

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.


Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Processamento Alternativo/genética , Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Éxons , Expressão Gênica , Humanos , NF-kappa B/metabolismo , Isoformas de Proteínas/genética , Precursores de RNA/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genética
7.
Nucleic Acids Res ; 48(15): 8349-8359, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621610

RESUMO

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.


Assuntos
Epigênese Genética , Processamento de Proteína Pós-Traducional/genética , Precursores de RNA/genética , Transcrição Genética , Animais , Diferenciação Celular/genética , Cromatina/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Genoma/genética , Código das Histonas/genética , Humanos , Regiões Promotoras Genéticas , Pseudogenes/genética , Precursores de RNA/metabolismo
8.
Signal Transduct Target Ther ; 5(1): 125, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661235

RESUMO

Stress proteins (SPs) including heat-shock proteins (HSPs), RNA chaperones, and ER associated stress proteins are molecular chaperones essential for cellular homeostasis. The major functions of HSPs include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. Regarded as a double-edged sword, HSPs also cooperate with numerous viruses and cancer cells to promote their survival. RNA chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnRNPs), which are essential factors for manipulating both the functions and metabolisms of pre-mRNAs/hnRNAs transcribed by RNA polymerase II. hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. Dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., Parkinson's diseases, Alzheimer disease), stroke and infectious diseases. In this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. As SPs also attract a great interest as potential antiviral targets (e.g., COVID-19), we also discuss the present progress and challenges in this area of HSP-based drug development, as well as with compounds already under clinical evaluation.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Proteínas de Choque Térmico/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Antivirais/síntese química , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/agonistas , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Terapia de Alvo Molecular/métodos , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Transcrição Genética/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
9.
Nucleic Acids Res ; 48(14): 7609-7622, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32476018

RESUMO

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.


Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Proteínas de Drosophila/fisiologia , Éxons , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fosfotransferases/metabolismo , Fosfotransferases/fisiologia , Clivagem do RNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
10.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561742

RESUMO

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Espectrometria de Massas , Mutação , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA-Seq , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
11.
Nat Commun ; 11(1): 2837, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503992

RESUMO

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.


Assuntos
Íntrons/genética , Precursores de RNA/metabolismo , Processamento de RNA , RNA Bacteriano/metabolismo , Spliceossomos/metabolismo , Bacillaceae/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA Bacteriano/genética , Análise Espaço-Temporal
12.
Science ; 369(6504): 656-663, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32586950

RESUMO

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.


Assuntos
Endorribonucleases/química , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Ribonucleases/química , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Holoenzimas/química , Conformação Proteica , RNA Catalítico/química , Especificidade por Substrato
13.
Nucleic Acids Res ; 48(W1): W268-W274, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32356893

RESUMO

Arm selection, the preferential expression of a 3' or 5' mature microRNA (miRNA), is a highly dynamic and tissue-specific process. Time-dependent expression shifts or switches between the arms are also relevant for human diseases. We present miRSwitch, a web server to facilitate the analysis and interpretation of arm selection events. Our species-independent tool evaluates pre-processed small non-coding RNA sequencing (sncRNA-seq) data, i.e. expression matrices or output files from miRNA quantification tools (miRDeep2, miRMaster, sRNAbench). miRSwitch highlights potential changes in the distribution of mature miRNAs from the same precursor. Group comparisons from one or several user-provided annotations (e.g. disease states) are possible. Results can be dynamically adjusted by choosing from a continuous range of highly specific to very sensitive parameters. Users can compare potential arm shifts in the provided data to a human reference map of pre-computed arm shift frequencies. We created this map from 46 tissues and 30 521 samples. As case studies we present novel arm shift information in a Alzheimer's disease biomarker data set and from a comparison of tissues in Homo sapiens and Mus musculus. In summary, miRSwitch offers a broad range of customized arm switch analyses along with comprehensive visualizations, and is freely available at: https://www.ccb.uni-saarland.de/mirswitch/.


Assuntos
MicroRNAs/metabolismo , Software , Doença de Alzheimer/genética , Animais , Humanos , Camundongos , MicroRNAs/química , Precursores de RNA/metabolismo , Análise de Sequência de RNA
15.
Nucleic Acids Res ; 48(11): 6294-6309, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402057

RESUMO

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.


Assuntos
Pareamento de Bases , Éxons , Íntrons , Processamento de RNA , Adenoviridae/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons/genética , Inativação Gênica , Íntrons/genética , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA/genética , Spliceossomos/metabolismo , Transcriptoma/genética , Globinas beta/genética
16.
PLoS One ; 15(4): e0231639, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324763

RESUMO

The anti-inflammatory cytokine interleukin-10 (IL10) is essential for attenuating inflammatory responses, which includes reducing the expression of pro-inflammatory microRNA-155 (miR155) in lipopolysaccharide (LPS) activated macrophages. miR155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SHIP1 and SOCS1. We previously found that IL10 interfered with the maturation of pre-miR155 to miR155. To understand the mechanism by which IL10 interferes with pre-miR155 maturation we isolated proteins that associate with pre-miR155 in response to IL10 in macrophages. We identified CELF2, a member of the CUGBP, ELAV-Like Family (CELF) family of RNA binding proteins, as protein whose association with pre-miR155 increased in IL10 treated cells. CRISPR-Cas9 mediated knockdown of CELF2 impaired IL10's ability to inhibit both miR155 expression and TNFα expression.


Assuntos
Proteínas CELF/metabolismo , Interleucina-10/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Animais , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , MicroRNAs/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Células RAW 264.7 , Precursores de RNA/genética , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/metabolismo
17.
Nucleic Acids Res ; 48(10): 5349-5365, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313933

RESUMO

Growing mammalian oocytes accumulate substantial amounts of RNA, most of which is degraded during subsequent meiotic maturation. The growth-to-maturation transition begins with germinal vesicle or nuclear envelope breakdown (GVBD) and is critical for oocyte quality and early development. The molecular machinery responsible for the oocyte transcriptome transition remains unclear. Here, we report that an exosome-associated RNase, EXOSC10, sculpts the transcriptome to facilitate the growth-to-maturation transition of mouse oocytes. We establish an oocyte-specific conditional knockout of Exosc10 in mice using CRISPR/Cas9 which results in female subfertility due to delayed GVBD. By performing multiple single oocyte RNA-seq, we document dysregulation of several types of RNA, and the mRNAs that encode proteins important for endomembrane trafficking and meiotic cell cycle. As expected, EXOSC10-depleted oocytes have impaired endomembrane components including endosomes, lysosomes, endoplasmic reticulum and Golgi. In addition, CDK1 fails to activate, possibly due to persistent WEE1 activity, which blocks lamina phosphorylation and disassembly. Moreover, we identified rRNA processing defects that cause higher percentage of developmentally incompetent oocytes after EXOSC10 depletion. Collectively, we propose that EXOSC10 promotes normal growth-to-maturation transition in mouse oocytes by sculpting the transcriptome to degrade RNAs encoding growth-phase factors and, thus, support the maturation phase of oogenesis.


Assuntos
Exorribonucleases/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese , Transcriptoma , Animais , Proteína Quinase CDC2/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Feminino , Infertilidade Feminina/genética , Membranas Intracelulares/metabolismo , Camundongos , Lâmina Nuclear/metabolismo , Poli A , RNA/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , RNA-Seq
18.
Nat Commun ; 11(1): 1518, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251279

RESUMO

Size selectivity is an important mechanism for molecular recognition based on the size difference between targets and non-targets. However, rational design of an artificial size-selective molecular recognition system for biological targets in living cells remains challenging. Herein, we construct a DNA molecular sieve for size-selective molecular recognition to improve the biosensing selectivity in living cells. The system consists of functional nucleic acid probes (e.g., DNAzymes, aptamers and molecular beacons) encapsulated into the inner cavity of framework nucleic acid. Thus, small target molecules are able to enter the cavity for efficient molecular recognition, while large molecules are prohibited. The system not only effectively protect probes from nuclease degradation and nonspecific proteins binding, but also successfully realize size-selective discrimination between mature microRNA and precursor microRNA in living cells. Therefore, the DNA molecular sieve provides a simple, general, efficient and controllable approach for size-selective molecular recognition in biomedical studies and clinical diagnoses.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Catalítico/química , Sondas Moleculares/química , Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/metabolismo , MicroRNAs/metabolismo , Sondas Moleculares/metabolismo , Tamanho da Partícula , Precursores de RNA/metabolismo , Especificidade por Substrato
19.
Nature ; 580(7801): 147-150, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238924

RESUMO

Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.


Assuntos
Cromatina/genética , Cromatina/metabolismo , RNA Longo não Codificante/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcrição Genética , Animais , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese , Motivos de Nucleotídeos , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Longo não Codificante/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo
20.
Biochem Biophys Res Commun ; 526(1): 261-266, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32209257

RESUMO

The last several years have seen exciting advances in the understanding of the structure and function of higher-order structures of RNA. Expression levels of some specific genes were shown to be directly regulated by environmentally-responsive formation of certain secondary structures such as stem-loops and pseudoknots. Even among these noncanonical structures, RNA G-quadruplexes, which form on the regions of guanine-rich sequences in mRNA, are highly stable structures that are involved in a variety of biological processes. However, many questions regarding the biological significance of RNA G-quadruplexes remain unsettled, mainly because it is difficult to locate the structures in mRNA. This review focuses on emerging methods that locate RNA G-quadruplexes in mRNA by computational and biochemical techniques. In addition, recent reports on the biological functions of RNA G-quadruplexes are also covered to highlight their various roles in cells, such as in regulating mRNA processing and translation.


Assuntos
Quadruplex G , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Processamento Alternativo/genética , Precursores de RNA/metabolismo , Transporte de RNA/genética , RNA Mensageiro/metabolismo , Transcrição Reversa/genética
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