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1.
Genes (Basel) ; 13(11)2022 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-36421841

RESUMO

Background: Up frameshift protein 1 (UPF1) is a key component of nonsense-mediated mRNA decay (NMD) of mRNA containing premature termination codons (PTCs). The dysregulation of UPF1 has been reported in various cancers. However, the expression profile of UPF1 and its clinical significance in clear cell renal cell carcinoma (ccRCC) remains unclear. Methods: In order to detect UPF1 expression in ccRCC and its relationship with the clinical features of ccRCC, bulk RNA sequencing data were analyzed from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and ArrayExpress databases. The impact of UPF1 on the immune microenvironment of ccRCC was evaluated by multiple immune scoring algorithms to identify the cell groups that typically express UPF1 using ccRCC single cell sequencing (scRNA) data. In addition, genes co-expressed with UPF1 were identified by the weighted gene correlation network analysis (WGCNA), followed by KEGG and Reactome enrichment analysis. A series of functional experiments were performed to assess the roles of UPF1 in renal cancer cells. Finally, pan-cancer analysis of UPF1 was also performed. Results: Compared with normal tissues, the expression levels of UPF1 mRNA and protein in tumor tissues of ccRCC patients decreased significantly. In addition, patients with low expression of UPF1 had a worse prognosis. Analysis of the immune microenvironment indicated that UPF1 immune cell infiltration was closely related and the ccRCC scRNA-seq data identified that UPF1 was mainly expressed in macrophages. WGCNA analysis suggested that the functions of co-expressed genes are mainly enriched in cell proliferation and cellular processes. Experimental tests showed that knockdown of UPF1 can promote the invasion, migration and proliferation of ccRCC cells. Lastly, pan-cancer analysis revealed that UPF1 disorders were closely associated with various cancer outcomes. Conclusions: UPF1 may play a tumor suppressive role in ccRCC and modulate the immune microenvironment. The loss of UPF1 can predict the prognosis of ccRCC, making it a promising biomarker and providing a new reference for prevention and treatment.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Prognóstico , RNA Helicases/genética , RNA Helicases/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Microambiente Tumoral/genética , Transativadores/genética , Transativadores/metabolismo
2.
Pharmacol Res ; 186: 106548, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36336216

RESUMO

Ras-GAP SH3 domain binding proteins (G3BPs) are a family of RNA-binding proteins that include G3BP1 and G3BP2 in mammals. The protein structure of G3BP2 allows it to bind with RNA or protein and regulate the nucleoplasmic shuttle, therefore participating in a variety of biological functions such as cell growth, differentiation and migration, and RNA and protein metabolism. G3BP2 is abnormally expressed in many tumors and diseases, such as breast cancer, lung cancer, prostate cancer, cardiac hypertrophy, and atherosclerosis. Many researchers have found that G3BP2 may be a potential therapeutic target. In this review, we examine the structure and expression regulation of G3BP2, and further summarize the function of G3BP2 from three aspects: RNA stabilization, protein subcellular localization, and stress granules assembling to provide a broader understanding of the role of G3BP2.


Assuntos
Neoplasias da Mama , DNA Helicases , Masculino , Animais , Humanos , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , DNA Helicases/genética , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias da Mama/metabolismo , RNA , Mamíferos/genética , Mamíferos/metabolismo , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
3.
Molecules ; 27(21)2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36364347

RESUMO

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Reposicionamento de Medicamentos , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , COVID-19/tratamento farmacológico , DNA Helicases/metabolismo , Antivirais/farmacologia
4.
RNA ; 28(12): 1621-1642, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36192133

RESUMO

Upf1, Upf2, and Upf3, the central regulators of nonsense-mediated mRNA decay (NMD), appear to exercise their NMD functions while bound to elongating ribosomes, and evidence for this conclusion is particularly compelling for Upf1. Hence, we used selective profiling of yeast Upf1:ribosome association to define that step in greater detail, understand whether the nature of the mRNA being translated influences Upf1:80S interaction, and elucidate the functions of ribosome-associated Upf1. Our approach has allowed us to clarify the timing and specificity of Upf1 association with translating ribosomes, obtain evidence for a Upf1 mRNA surveillance function that precedes the activation of NMD, identify a unique ribosome state that generates 37-43 nt ribosome footprints whose accumulation is dependent on Upf1's ATPase activity, and demonstrate that a mutated form of Upf1 can interfere with normal translation termination and ribosome release. In addition, our results strongly support the existence of at least two distinct functional Upf1 complexes in the NMD pathway.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases , RNA Helicases/genética , RNA Helicases/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Med Mycol ; 60(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36240494

RESUMO

The yeast SKI (superkiller) complex was originally identified from cells that were infected by the M 'killer' virus. Ski2, as the core of the SKI complex, is a cytoplasmic cofactor and regulator of RNA-degrading exosome. The putative RNA helicase Ski2 was highly conserved from yeast to animals and has been demonstrated to play a key role in the regulation of RNA surveillance, temperature sensitivity, and growth in several yeasts but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Ski2 protein, named SKI2, in the fungal pathogen C. neoformans. To obtain insights into the function of Ski2, we created a mutant strain, ski2Δ, with the CRISPR-Cas9 editing tool. Disruption of SKI2 impaired cell wall integrity. Further investigations revealed the defects of the ski2Δ mutant in resistance to osmotic stresses and extreme growth temperatures. However, significantly, the ability to undergo invasive growth under nutrient-depleted conditions was increased in the ski2Δ mutant. More importantly, our results showed that the ski2Δ mutant exhibited slightly lower virulence and severe susceptibility to anti-ribosomal drugs by comparison to the wild type, but it developed multidrug resistance to azoles and flucytosine. By constructing the double deletion strain ski2Δafr1Δ, we verified that increased Afr1 in ski2Δ contributed to the azole resistance, which might be influenced by nonclassical small interfering RNA. Our work suggests that Ski2 plays critical roles in drug resistance and regulation of gene transcription in the yeast pathogen C. neoformans.


Assuntos
Cryptococcus neoformans , Farmacorresistência Fúngica , Proteínas Fúngicas , Azóis/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , RNA Helicases/metabolismo , Farmacorresistência Fúngica/genética , Estresse Fisiológico
6.
Proc Natl Acad Sci U S A ; 119(43): e2205255119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36256816

RESUMO

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid-liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect-based chemical-enrichment method (SECEM) coupled with liquid chromatography-tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG-rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG-rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.


Assuntos
Arginina , Grânulos Citoplasmáticos , Animais , Humanos , Arginina/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteoma/metabolismo , Mamíferos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
7.
J Zhejiang Univ Sci B ; 23(10): 863-875, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36226539

RESUMO

Up-frameshift 1 (UPF1), as the most critical factor in nonsense-mediated messenger RNA (mRNA) decay (NMD), regulates tumor-associated molecular pathways in many cancers. However, the role of UPF1 in lung adenocarcinoma (LUAD) amino acid metabolism remains largely unknown. In this study, we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics. We further confirmed that UPF1 knockdown inhibited activating transcription factor 4 (ATF4) and Ser51 phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), the core proteins in amino acid metabolism reprogramming. In addition, UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells, which depends on the function of ATF4. Clinically, UPF1 mRNA expression is abnormal in LUAD tissues, and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival (OS) in LUAD patients. Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Fator 4 Ativador da Transcrição/genética , Aminoácidos , Proliferação de Células , Fator de Iniciação 2 em Eucariotos , Humanos , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismo
8.
Proc Natl Acad Sci U S A ; 119(44): e2207975119, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36279435

RESUMO

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.


Assuntos
DNA Helicases , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , DNA Helicases/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos de Estresse , Proteínas Ativadoras de GTPase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
9.
BMC Biol ; 20(1): 194, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050755

RESUMO

BACKGROUND: Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. RESULTS: Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. CONCLUSIONS: These findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA , Regiões 3' não Traduzidas , Proteínas Argonauta/genética , Humanos , Hipóxia/genética , MicroRNAs/metabolismo , Proteínas do Fator Nuclear 90/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Front Immunol ; 13: 985792, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36059486

RESUMO

Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.


Assuntos
Poli I-C , RNA Helicases , Peixe-Zebra , Animais , Antivirais/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Interferons , Mamíferos/metabolismo , Poli I-C/farmacologia , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
11.
PLoS Pathog ; 18(9): e1010743, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36067236

RESUMO

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.


Assuntos
Antivirais , DNA Helicases , Antivirais/metabolismo , DNA Helicases/metabolismo , Interferons/metabolismo , Nucleosídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
12.
Inorg Chem ; 61(39): 15664-15677, 2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36125417

RESUMO

The identification of novel therapeutics against the pandemic SARS-CoV-2 infection is an indispensable new address of current scientific research. In the search for anti-SARS-CoV-2 agents as alternatives to the vaccine or immune therapeutics whose efficacy naturally degrades with the occurrence of new variants, the salts of Bi3+ have been found to decrease the activity of the Zn2+-dependent non-structural protein 13 (nsp13) helicase, a key component of the SARS-CoV-2 molecular tool kit. Here, we present a multilevel computational investigation based on the articulation of DFT calculations, classical MD simulations, and MIF analyses, focused on the examination of the effects of Bi3+/Zn2+ exchange on the structure and molecular interaction features of the nsp13 protein. Our calculations confirmed that Bi3+ ions can replace Zn2+ in the zinc-finger metal centers and cause slight but appreciable structural modifications in the zinc-binding domain of nsp13. Nevertheless, by employing an in-house-developed ATOMIF tool, we evidenced that such a Bi3+/Zn2+ exchange may decrease the extension of a specific hydrophobic portion of nsp13, responsible for the interaction with the nsp12 protein. The present study provides for a detailed, atomistic insight into the potential anti-SARS-CoV-2 activity of Bi3+ and, more generally, evidences the hampering of the nsp13-nsp12 interaction as a plausible therapeutic strategy.


Assuntos
COVID-19 , SARS-CoV-2 , Bismuto , Humanos , Íons , RNA Helicases/química , RNA Helicases/metabolismo , Sais , Zinco
13.
J Biol Chem ; 298(11): 102486, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36108740

RESUMO

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.


Assuntos
Hepatite C , Neoplasias Hepáticas , Humanos , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Hepatite C/metabolismo , RNA Helicases/metabolismo
14.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36179369

RESUMO

We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , DNA Helicases , RNA Helicases , Animais , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos de Estresse , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
15.
Proc Natl Acad Sci U S A ; 119(38): e2205842119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095196

RESUMO

RNA uridylation, catalyzed by terminal uridylyl transferases (TUTases), represents a conserved and widespread posttranscriptional RNA modification in eukaryotes that affects RNA metabolism. In plants, several TUTases, including HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1), have been characterized through genetic and biochemical approaches. However, little is known about their physiological significance during plant development. Here, we show that HESO1 and URT1 act cooperatively with the cytoplasmic 3'-5' exoribonucleolytic machinery component SUPERKILLER 2 (SKI2) to regulate photosynthesis through RNA surveillance of the Calvin cycle gene TRANSKETOLASE 1 (TKL1) in Arabidopsis. Simultaneous dysfunction of HESO1, URT1, and SKI2 resulted in leaf etiolation and reduced photosynthetic efficiency. In addition, we detected massive illegitimate short interfering RNAs (siRNAs) from the TKL1 locus in heso1 urt1 ski2, accompanied by reduced TKL1/2 expression and attenuated TKL activities. Consequently, the metabolic analysis revealed that the abundance of many Calvin cycle intermediates is dramatically disturbed in heso1 urt1 ski2. Importantly, all these molecular and physiological defects were largely rescued by the loss-of-function mutation in RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), demonstrating illegitimate siRNA-mediated TKL silencing. Taken together, our results suggest that HESO1- and URT1-mediated RNA uridylation connects to the cytoplasmic RNA degradation pathway for RNA surveillance, which is crucial for TKL expression and photosynthesis in Arabidopsis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fotossíntese , RNA Nucleotidiltransferases , Estabilidade de RNA , RNA Interferente Pequeno , Transcetolase , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleotidiltransferases/metabolismo , Fotossíntese/genética , RNA Helicases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcetolase/genética , Transcetolase/metabolismo , Uridina/metabolismo
16.
Viruses ; 14(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36146752

RESUMO

Prions are infectious proteins, mostly having a self-propagating amyloid (filamentous protein polymer) structure consisting of an abnormal form of a normally soluble protein. These prions arise spontaneously in the cell without known reason, and their effects were generally considered to be fatal based on prion diseases in humans or mammals. However, the wide array of prion studies in yeast including filamentous fungi revealed that their effects can range widely, from lethal to very mild (even cryptic) or functional, depending on the nature of the prion protein and the specific prion variant (or strain) made by the same prion protein but with a different conformation. This prion biology is affected by an array of molecular chaperone systems, such as Hsp40, Hsp70, Hsp104, and combinations of them. In parallel with the systems required for prion propagation, yeast has multiple anti-prion systems, constantly working in the normal cell without overproduction of or a deficiency in any protein, which have negative effects on prions by blocking their formation, curing many prions after they arise, preventing prion infections, and reducing the cytotoxicity produced by prions. From the protectors of nascent polypeptides (Ssb1/2p, Zuo1p, and Ssz1p) to the protein sequesterase (Btn2p), the disaggregator (Hsp104), and the mysterious Cur1p, normal levels of each can cure the prion variants arising in its absence. The controllers of mRNA quality, nonsense-mediated mRNA decay proteins (Upf1, 2, 3), can cure newly formed prion variants by association with a prion-forming protein. The regulator of the inositol pyrophosphate metabolic pathway (Siw14p) cures certain prion variants by lowering the levels of certain organic compounds. Some of these proteins have other cellular functions (e.g., Btn2), while others produce an anti-prion effect through their primary role in the normal cell (e.g., ribosomal chaperones). Thus, these anti-prion actions are the innate defense strategy against prions. Here, we outline the anti-prion systems in yeast that produce innate immunity to prions by a multi-layered operation targeting each step of prion development.


Assuntos
Príons , Proteínas de Saccharomyces cerevisiae , Difosfatos/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Inositol/metabolismo , Chaperonas Moleculares/metabolismo , Polímeros/metabolismo , Proteínas Priônicas/metabolismo , Príons/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo
17.
Viruses ; 14(8)2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-36016333

RESUMO

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b-NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.


Assuntos
Vírus da Dengue , DNA Helicases/metabolismo , Vírus da Dengue/genética , Simulação de Acoplamento Molecular , RNA/metabolismo , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral
18.
Sci Adv ; 8(34): eabq2945, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-36001654

RESUMO

Meiosis entry during spermatogenesis requires reprogramming from mitotic to meiotic gene expression profiles. Transcriptional regulation has been extensively studied in meiosis entry, but gain of function for master transcription factors is insufficient to down-regulate mitotic genes. RNA helicase YTHDC2 and its partner MEIOC emerge as essential posttranscriptional regulators of meiotic entry. However, it is unclear what governs the RNA binding specificity of YTHDC2/MEIOC. Here, we identified RNA binding protein RBM46 as a component of the YTHDC2/MEIOC complex. Testis-specific Rbm46 knockout in mice causes infertility with defective mitotic-to-meiotic transition, phenocopying global Ythdc2 or Meioc knockout. RBM46 binds to 3' UTR of mitotic transcripts within 100 nucleotides from YTHDC2 U-rich motifs and targets these transcripts for degradation. Dysregulated RBM46 expression is associated with human male fertility disorders. These findings establish the RBM46/YTHDC2/MEIOC complex as the major posttranscriptional regulator responsible for down-regulating mitotic transcripts during meiosis entry in mammalian spermatogenesis, with implications for understanding meiosis-related fertility disorders.


Assuntos
Meiose , Proteínas de Ligação a RNA/metabolismo , Espermatogênese , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Masculino , Mamíferos/metabolismo , Meiose/genética , Camundongos , Camundongos Knockout , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Espermatogênese/genética , Testículo/metabolismo
19.
J Exp Clin Cancer Res ; 41(1): 252, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35986402

RESUMO

BACKGROUND: The accumulating evidence confirms that long non-coding RNAs (lncRNAs) play a critical regulatory role in the progression of renal cell carcinoma (RCC). But, the application of lncRNAs in gene therapy remains scarce. Here, we investigated the efficacy of a delivery system by introducing the plasmid-encoding tumor suppressor lncRNA-SLERCC (SLERCC) in RCC cells. METHODS: We performed lncRNAs expression profiling in paired cancer and normal tissues through microarray and validated in our clinical data and TCGA dataset. The Plasmid-SLERCC@PDA@MUC12 nanoparticles (PSPM-NPs) were tested in vivo and in vitro, including cellular uptake, entry, CCK-8 assay, tumor growth inhibition, histological assessment, and safety evaluations. Furthermore, experiments with nude mice xenografts model were performed to evaluate the therapeutic effect of PSPM-NPs nanotherapeutic system specific to the SLERCC. RESULTS: We found that the expression of SLERCC was downregulated in RCC tissues, and exogenous upregulation of SLERCC could suppress metastasis of RCC cells. Furthermore, high expression DNMT3A was recruited at the SLERCC promoter, which induced aberrant hypermethylation, eventually leading to downregulation of SLERCC expression in RCC. Mechanistically, SLERCC could directly bind to UPF1 and exert tumor-suppressive effects through the Wnt/ß-catenin signaling pathway, thereby inhibiting progression and metastasis in RCC. Subsequently, the PSPM-NPs nanotherapeutic system can effectively inhibit the growth of RCC metastases in vivo. CONCLUSIONS: Our findings suggested that SLERCC is a promising therapeutic target and that plasmid-encapsulated nanomaterials targeting transmembrane metastasis markers may open a new avenue for the treatment in RCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Nanopartículas , RNA Longo não Codificante , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/terapia , Camundongos , Camundongos Nus , Plasmídeos/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transativadores/metabolismo , Transfecção
20.
J Biol Chem ; 298(10): 102383, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35987382

RESUMO

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.


Assuntos
Adenosina Trifosfatases , DNA de Cadeia Simples , Vírus da Encefalite Transmitidos por Carrapatos , RNA Helicases , Proteínas não Estruturais Virais , Humanos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/enzimologia , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Fosfatos/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas não Estruturais Virais/metabolismo
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