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1.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203408

RESUMO

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Mutação/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Neoplasias do Endométrio/genética , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Células MCF-7 , Reação em Cadeia da Polimerase , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
2.
Aging (Albany NY) ; 13(12): 15750-15769, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34102611

RESUMO

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated ß-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.


Assuntos
Ceramidase Ácida/metabolismo , Senescência Celular , Fibroblastos/citologia , Fibroblastos/enzimologia , Ceramidase Ácida/genética , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular , Ceramidas/metabolismo , Inativação Gênica , Humanos , Metaboloma , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética
3.
Mol Cell ; 81(14): 2901-2913.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34157309

RESUMO

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , Domínio Catalítico/genética , Endorribonucleases/genética , Exorribonucleases/genética , Regulação Bacteriana da Expressão Gênica/genética , Chaperonas Moleculares/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genética
4.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066757

RESUMO

Despite advances in diagnostic, prognostic, and treatment modalities, myocardial infarction (MI) remains a leading cause of morbidity and mortality. Impaired cellular signaling after an MI causes maladaptive changes resulting in cardiac remodeling. MicroRNAs (miRNAs/miR) along with other molecular components have been investigated for their involvement in cellular signaling in the pathogenesis of various cardiac conditions like MI. miRNAs are small non-coding RNAs that negatively regulate gene expression. They bind to complementary mRNAs and regulate the rate of protein synthesis by altering the stability of their targeted mRNAs. A single miRNA can modulate several cellular signaling pathways by targeting hundreds of mRNAs. This review focuses on the biogenesis and beneficial effects of cellular and circulating (exosomal) miRNAs on cardiac remodeling after an MI. Particularly, miR-1, -133, 135, and -29 that play an essential role in cardiac remodeling after an MI are described in detail. The limitations that will need to be addressed in the future for the further development of miRNA-based therapeutics for cardiovascular conditions will also be discussed.


Assuntos
MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Infarto do Miocárdio/sangue , Infarto do Miocárdio/fisiopatologia , Remodelação Ventricular , Animais , Biomarcadores/sangue , MicroRNA Circulante/uso terapêutico , Humanos , Modelos Biológicos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/genética , Estabilidade de RNA/genética
5.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073862

RESUMO

Heat stress is a major limiting factor of grain yield and quality in crops. Abiotic stresses have a transgenerational impact and the mechanistic basis is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis heat stress in tetraploid wheat. Leaf physiological traits, harvest components and grain quality traits were characterized under the impact of parental and progeny heat stress. The parental heat stress treatment had a positive influence on the offspring for traits including chlorophyll content, grain weight, grain number and grain total starch content. Integrated sequencing analysis of the small RNAome, mRNA transcriptome and degradome provided the first description of the molecular networks mediating heat stress adaptation under transgenerational influence. The expression profile of 1771 microRNAs (733 being novel) and 66,559 genes was provided, with differentially expressed microRNAs and genes characterized subject to the progeny treatment, parental treatment and tissue-type factors. Gene Ontology and KEGG pathway analysis of stress responsive microRNAs-mRNA modules provided further information on their functional roles in biological processes such as hormone homeostasis, signal transduction and protein stabilization. Our results provide new insights on the molecular basis of transgenerational heat stress adaptation, which can be used for improving thermo-tolerance in breeding.


Assuntos
Adaptação Fisiológica/genética , Regulação da Expressão Gênica de Plantas/genética , Resposta ao Choque Térmico/genética , MicroRNAs/metabolismo , Transcriptoma/genética , Clorofila/análise , Regulação para Baixo , Perfilação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Folhas de Planta/metabolismo , Estabilidade de RNA/genética , Sementes/metabolismo , Amido/análise , Tetraploidia , Triticum/genética , Regulação para Cima
6.
J Biol Chem ; 297(1): 100816, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023389

RESUMO

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.


Assuntos
Mitocôndrias/metabolismo , RNA de Transferência de Alanina/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Sequência de Bases , Citocromos c/metabolismo , Transporte de Elétrons , Humanos , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Mutação/genética , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mitocondrial/genética , RNA de Transferência de Alanina/química , Espécies Reativas de Oxigênio/metabolismo , Aminoacilação de RNA de Transferência
7.
Biochimie ; 187: 67-74, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022290

RESUMO

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.


Assuntos
Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética
8.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799331

RESUMO

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5'tRNA-derived stress-induced RNAs (5'tiRNAs), namely 5'tiRNA-GlyGCC and 5'tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.


Assuntos
Estresse Oxidativo/genética , RNA de Transferência/genética , Ribonuclease Pancreático/genética , tRNA Metiltransferases/genética , Humanos , Clivagem do RNA/genética , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , Estresse Fisiológico/genética , Uridina/análogos & derivados , Uridina/genética
9.
Mol Genet Metab ; 133(2): 137-147, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33795191

RESUMO

Tristetraprolin (TTP) is a nucleocytoplasmic 326 amino acid protein whose sequence is characterized by possessing two CCCH-type zinc finger domains. In the cytoplasm TTP function is to promote the degradation of mRNAs that contain adenylate/uridylate-rich elements (AREs). Mechanistically, TTP promotes the recruitment of poly(A)-specific deadenylases and exoribonucleases. By reducing the half-life of about 10% of all the transcripts in the cell TTP has been shown to participate in multiple cell processes that include regulation of gene expression, cell proliferation, metabolic homeostasis and control of inflammation and immune responses. However, beyond its role in mRNA decay, in the cell nucleus TTP acts as a transcriptional coregulator by interacting with chromatin modifying enzymes. TTP has been shown to repress the transactivation of NF-κB and estrogen receptor suggesting the possibility that it participates in the transcriptional regulation of hundreds of genes in human cells and its possible involvement in breast cancer progression. In this review, we discuss the cytoplasmic and nuclear functions of TTP and the effect of the dysregulation of its protein levels in the development of human diseases. We suggest that TTP be classified as a moonlighting tumor supressor protein that regulates gene expression through two different mechanims; the decay of ARE-mRNAs and a transcriptional coregulatory function.


Assuntos
Citosol/metabolismo , RNA Mensageiro/metabolismo , Ativação Transcricional/genética , Tristetraprolina/genética , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/patologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , Tristetraprolina/metabolismo , Dedos de Zinco/genética
10.
J Virol ; 95(13): e0009721, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33883220

RESUMO

YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A)-modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A methylated, and this modification regulates the viral life cycle. HBV is particularly interesting, as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it positively affects pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the virus life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP- or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate in the nucleus to reduce reverse transcription in HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Adenosina/análogos & derivados , DNA Viral/química , Proteína do X Frágil de Retardo Mental/metabolismo , Vírus da Hepatite B/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Processamento de RNA/metabolismo , Adenosina/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Replicação do DNA/genética , Proteína do X Frágil de Retardo Mental/genética , Regulação Viral da Expressão Gênica/genética , Humanos , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Estabilidade de RNA/genética , Transcrição Genética/genética , Replicação Viral/genética
11.
PLoS Pathog ; 17(4): e1009530, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909701

RESUMO

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.


Assuntos
Adenosina/análogos & derivados , RNA Helicases DEAD-box/fisiologia , Evasão da Resposta Imune/genética , Estabilidade de RNA/genética , Viroses , Adenosina/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Cricetinae , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Replicação Viral/genética
12.
Nucleic Acids Res ; 49(8): 4456-4471, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823555

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. However, regulation of viral miRNAs remains largely unknown. Our prior studies demonstrated that MCPIP1 regulates KSHV miRNA biogenesis by degrading most KSHV pre-miRNAs through its RNase activity. Some viral pre-miRNAs are partially resistant to degradation by MCPIP1. Here, we further characterized MCPIP1 substrate specificity and its antiviral potential against KSHV infection. In vitro cleavage assays and binding assays showed that MCPIP1 cleavage efficiency is related to binding affinity. Motif-based sequence analysis identified that KSHV pre-miRNAs that are well degraded by MCPIP1 have a 5-base motif (M5 base motif) within their terminal loops and this motif region consists of multiple pyrimidine-purine-pyrimidine (YRY) motifs. We further demonstrated that mutation of this M5 base motif within terminal loop of pre-miRNAs inhibited MCPIP1-mediated RNA degradation. We also revealed that MCPIP1 has an antiviral effect against KSHV infection. MCPIP1 can reduce the expression of Dicer, which in turn restricts KSHV infection. Conclusively, our findings demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering the expression of miRNA biogenesis factors.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , MicroRNAs/genética , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA/genética , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
13.
Nat Commun ; 12(1): 2316, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875662

RESUMO

Synthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.


Assuntos
Autofagia/genética , Estabilidade de RNA/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Vacúolos/genética , Northern Blotting , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
14.
Plant Mol Biol ; 106(3): 271-284, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33864582

RESUMO

To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3'-5' and/or XRN4 5'-3' exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome-mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.


Assuntos
Exonucleases/metabolismo , Exossomos , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Interferência de RNA , Tabaco/metabolismo , Proteínas de Arabidopsis/genética , Códon de Iniciação/genética , Exonucleases/genética , Regulação da Expressão Gênica de Plantas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fases de Leitura Aberta , Proteínas de Plantas/genética , Estabilidade de RNA/genética , RNA de Plantas/genética , RNA Interferente Pequeno , Tabaco/genética
15.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649230

RESUMO

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.


Assuntos
Endorribonucleases , Splicing de RNA/genética , Estabilidade de RNA/genética , RNA Fúngico , RNA Mensageiro , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Exp Cell Res ; 401(2): 112524, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33651996

RESUMO

N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Ciclina D1/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Adenosina/análogos & derivados , Adenosina/genética , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Fase G1/genética , Humanos , Mioblastos/metabolismo
17.
Nat Commun ; 12(1): 1790, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741984

RESUMO

RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3' end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3'-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m6A writer complex, and m6A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromatina/genética , Flores/genética , Inativação Gênica , Proteínas de Domínio MADS/genética , Estruturas R-Loop , Proteínas de Ligação a RNA/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/metabolismo , Poliadenilação , Ligação Proteica , Estabilidade de RNA/genética , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
18.
Mol Biol Rep ; 48(3): 2315-2324, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33788053

RESUMO

Matrix-metalloproteinase-2 (MMP2) is a foremost MMP, governing invasion of breast cancer cells during metastasis. miR-20a was reported to induce mesenchymal to epithelial transition in MDA-MB-231 cells and its endogenous expression varies directly with invasiveness of breast cancer cells. The inverse and direct correlation of invasiveness with miR-20a and Nucleolin respectively led us to study the post-transcriptional regulation of MMP2 by miR-20a and mRNA stabilizing protein, Nucleolin. Thus, understanding the mechanism of its regulation will enable modification of the invasion potential. MMP2 was found to be higher in MDA-MB-231 than MCF-7 cells both at RNA and protein levels. RNA-protein co-immunoprecipitation assay with Argonaute 2 revealed that MMP2 undergoes miRNA-mediated post-transcriptional regulation. miR-20a decreased MMP2 expression as well as its enzymatic activity as found by zymogram assay. Reporter assay showed that miR-20a directly binds to its putative binding site in MMP2 3'-UTR as per in silico prediction. miR-20a additionally impeded MMP2 mRNA stability, and binding of stabilizing trans-factor Nucleolin to its 3'-UTR was confirmed by RNA-protein co-immunoprecipitation assay. Partial down-regulation of Nucleolin by Si-RNA resulted in the downregulation of MMP2 and Nucleolin over-expression rescued the inhibitory effect of miR-20a on MMP2 expression. Delineating the mechanism of post-transcriptional regulation of MMP2, two of its potent regulators, miR-20a and Nucleolin were identified. It was established for the first time that MMP2 is a direct target of miR-20a. The results also elucidated that Nucleolin binds to MMP2 3' UTR and its abundance affects MMP2 expression.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , MicroRNAs/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Simulação por Computador , Regulação para Baixo/genética , Feminino , Humanos , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/genética , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Biochem Biophys Res Commun ; 548: 134-142, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33640606

RESUMO

Sleeve gastrectomy (SG) is the most widely used bariatric procedures globally, which could improve glucose and lipid metabolism dramatically. Circular RNAs (circRNAs) are being increasingly implicated in numerous pathophysiological processes. However, for diabetes mellitus (DM), the expression and function of circRNAs remain largely undetermined, in particular, whether circRNAs mediate the amelioration of DM observed after SG. Using a diabetic rat model, we subjected liver tissue from SG and sham-operated rats to RNA sequencing. Amongst the 103 differentially regulated circRNAs identified in diabetic rats after SG, we focused on circDOCK7, a highly expressed circRNA derived from the back-splicing of the DOCK7 gene. Silencing of circDOCK7 significantly inhibited cellular proliferation and induction of apoptosis in insulin-resistant rat hepatocytes. Further analysis indicated circDOCK7 harbored binding sites for miR-139-3p and regulated the expression of minichromosome maintenance 3 (MCM3) through sequestration of miR-139-3p. Our findings therefore demonstrate a novel regulatory pathway involving circDOCK7 that regulates cellular proliferation and apoptosis through increasing the expression of MCM3. Overall, our study establishes a list of specific circRNAs expressed in diabetic rat liver after SG including circDOCK7 which serve as potential biomarkers and treatment targets for DM patients.


Assuntos
Apoptose/genética , Diabetes Mellitus Experimental/genética , Regulação para Baixo/genética , Gastrectomia , Hepatócitos/patologia , MicroRNAs/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , RNA Circular/metabolismo , Animais , Sequência de Bases , Peso Corporal , Linhagem Celular , Proliferação de Células/genética , Diabetes Mellitus Experimental/patologia , Comportamento Alimentar , Inativação Gênica , Glucose/metabolismo , Hepatócitos/metabolismo , Homeostase , Masculino , MicroRNAs/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Estabilidade de RNA/genética , RNA Circular/genética , Ratos Wistar
20.
FEBS J ; 288(11): 3418-3423, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33590687

RESUMO

mRNA degradation rate is one of the key stages of gene expression regulation in eukaryotic cells. To date, intertwined processes of post-transcriptional control have been widely investigated, but focused rather on the examination of mechanisms controlling stability of particular protein-coding transcripts. Currently, a wealth of information from structural, biochemical, and high-throughput studies makes it tempting to define general rules governing mRNA stability that could be considered as versatile and valid on a genome-wide scale. Basu et al. analyzed multiple experimental and computational data on Saccharomyces cerevisiae mRNA half-lives as well as on secondary structures and protein-binding sites within transcripts, and collated it with available structures of ribonucleases, that is, enzymes responsible for mRNA degradation. This approach allowed to conclude how particular mRNA features such as lengths of unstructured terminal or internal regions or sequestration into ribonucleoprotein complexes impact half-lives of protein-coding transcripts and to define genome-scale principles of mRNA stability control in yeast.


Assuntos
Genoma Fúngico/genética , Estabilidade de RNA/genética , Saccharomyces cerevisiae/genética , Transcriptoma/genética , Regulação Fúngica da Expressão Gênica/genética , Ligação Proteica/genética , RNA Mensageiro/genética
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