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1.
Methods Enzymol ; 658: 1-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517943

RESUMO

Knowledge of the structural information is essential for understanding the functional details of modified RNA. Cellular non-coding RNA such as rRNA, tRNA and even viral RNAs contain a number of post-transcriptional modifications with varied degree of diversity and density. In this chapter, we discuss the use of a combination of biochemical and analytical tools such as ribonucleases and liquid chromatography coupled with mass spectrometry approaches for characterization of modified RNA. We present the protocols and alternate strategies for obtaining confident modified sequence information to facilitate the understanding of function.


Assuntos
RNA , Ribonucleases , Sequência de Bases , Cromatografia Líquida , RNA/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem
2.
Nat Commun ; 12(1): 5033, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413302

RESUMO

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.


Assuntos
Nucleotídeos de Adenina/química , COVID-19/diagnóstico , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , RNA Bacteriano/genética , Ribonucleases/metabolismo , SARS-CoV-2/genética , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade
3.
Arch Virol ; 166(10): 2711-2722, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34313859

RESUMO

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.


Assuntos
Fusarium/virologia , Genoma Viral/genética , Vírus de RNA de Cadeia Positiva/genética , Micovírus/classificação , Micovírus/genética , Micovírus/isolamento & purificação , Filogenia , Doenças das Plantas/microbiologia , Vírus de RNA de Cadeia Positiva/classificação , Vírus de RNA de Cadeia Positiva/isolamento & purificação , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Ribonucleases/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Esporos Fúngicos/virologia , Proteínas Virais/genética
4.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298861

RESUMO

The pathogenesis of hidradenitis suppurativa (HS) is yet to be fully understood. However, inflammation is a key element in the development of skin lesions. The aim of this study was to evaluate the expression of monocyte chemotactic protein-1-induced protein-1 (MCPIP1) in the skin of patients suffering from HS. Skin biopsies of 15 patients with HS and 15 healthy controls were obtained and processed for immunohistochemistry, western blot, and real time PCR. The highest mean MCPIP1 mRNA expression was found in the inflammatory lesional skin of HS patients. It was significantly higher than MCPIP1 mRNA expression in the biopsies from both healthy controls and non-lesional skin of HS patients. Western blot analysis indicated that expression of MCPIP1 was elevated within both lesional and non-lesional skin compared to the healthy control. The increased MCPIP1 mRNA and protein expression level in HS lesions may indicate its possible role in the disease pathogenesis.


Assuntos
Hidradenite Supurativa/metabolismo , Queratinócitos/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica/métodos , Inflamação/metabolismo , Masculino , RNA Mensageiro/metabolismo , Pele/metabolismo
5.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298932

RESUMO

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1ß mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.


Assuntos
Psoríase/metabolismo , Psoríase/patologia , Ribonucleases/metabolismo , Adulto , Animais , Células Cultivadas , Citocinas/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , RNA Mensageiro/genética , Pele/metabolismo , Pele/patologia
6.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299186

RESUMO

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O2) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs' survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.


Assuntos
Hipóxia Celular/fisiologia , Células Dendríticas/metabolismo , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Humanos , Monócitos/imunologia , Monócitos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ribonucleases/biossíntese , Ribonucleases/imunologia , Transdução de Sinais , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/imunologia
7.
Nat Commun ; 12(1): 4105, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215755

RESUMO

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Dermatite/metabolismo , Endorribonucleases/metabolismo , Inflamação/metabolismo , Células Mieloides/metabolismo , Ribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CCL2 , Células Dendríticas , Endorribonucleases/deficiência , Endorribonucleases/genética , Epigenômica , Humanos , Imiquimode , Inflamação/patologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Psoríase , Ribonucleases/deficiência , Ribonucleases/genética , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
8.
Science ; 372(6547): 1220-1224, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112695

RESUMO

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsídeo/metabolismo , Evolução Molecular Direcionada , RNA Mensageiro/metabolismo , Substituição de Aminoácidos , Aquifex/enzimologia , Proteínas de Bactérias/química , Capsídeo/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonucleases/metabolismo
9.
Methods Mol Biol ; 2323: 13-23, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086270

RESUMO

RNA is a pivotal element of the cell which is most of the time found in complex with protein(s) in a cellular environment. RNA can adopt three-dimensional structures that may form specific binding sites not only for proteins but for all sorts of molecules. Since the early days of molecular biology, strategies to probe RNA structure have been developed. Such probes are small molecules or RNases that most of the time specifically react with single strand nucleotides. The precise reaction or cleavage site can be mapped by reverse transcription. It appears that nucleotides in close contact or in proximity of a ligand are no longer reactive to these probes. Carrying the RNA probing experiment in parallel in presence and absence of a ligand yield differences that are known as the ligand "footprint." Such footprints allow for the identification of the precise site of the ligand interaction, but also reveals RNA structural rearrangement upon ligand binding. Here we provide an experimental and analytical workflow to carry RNA footprinting experiments.


Assuntos
Biologia Computacional/métodos , Técnicas de Sonda Molecular/instrumentação , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Conformação de Ácido Nucleico , Ribonucleases/metabolismo
10.
Nucleic Acids Res ; 49(12): 7164-7178, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139012

RESUMO

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.


Assuntos
Proteínas de Escherichia coli/química , Ribonucleases/química , Dimerização , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleases/genética , Ribonucleases/metabolismo , Especificidade por Substrato
11.
Nat Commun ; 12(1): 3878, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188032

RESUMO

Different levels of regulatory mechanisms, including posttranscriptional regulation, are needed to elaborately regulate inflammatory responses to prevent harmful effects. Terminal uridyltransferase 7 (TUT7) controls RNA stability by adding uridines to its 3' ends, but its function in innate immune response remains obscure. Here we reveal that TLR4 activation induces TUT7, which in turn selectively regulates the production of a subset of cytokines, including Interleukin 6 (IL-6). TUT7 regulates IL-6 expression by controlling ribonuclease Regnase-1 mRNA (encoded by Zc3h12a gene) stability. Mechanistically, TLR4 activation causes TUT7 to bind directly to the stem-loop structure on Zc3h12a 3'-UTR, thereby promotes Zc3h12a uridylation and degradation. Zc3h12a from LPS-treated TUT7-sufficient macrophages possesses increased oligo-uridylated ends with shorter poly(A) tails, whereas oligo-uridylated Zc3h12a is significantly reduced in Tut7-/- cells after TLR4 activation. Together, our findings reveal the functional role of TUT7 in sculpting TLR4-driven responses by modulating mRNA stability of a selected set of inflammatory mediators.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/genética , Receptor 4 Toll-Like/metabolismo , Regiões 3' não Traduzidas , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Estabilidade de RNA , RNA Mensageiro/genética , Ribonucleases/metabolismo , Uridina Monofosfato/metabolismo
12.
J Biol Chem ; 296: 100781, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34000298

RESUMO

The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1α is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1α dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1α, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix αK, located between the kinase and RNase domains of IRE1α, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1α differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix αK is a conduit for the activation of IRE1α in response to stress.


Assuntos
Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular , Endorribonucleases/química , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Ribonucleases/metabolismo
13.
Nucleic Acids Res ; 49(10): 5798-5812, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037799

RESUMO

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Alinhamento de Sequência
14.
Nucleic Acids Res ; 49(9): 5369-5381, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33950203

RESUMO

The CCCH-type zinc finger (ZnF) containing ZC3H12 ribonucleases are crucial in post-transcriptional immune homoeostasis with ZC3H12A being the only structurally studied member of the family. In this study, we present a structural-biochemical characterization of ZC3H12C, which is linked with chronic immune disorders like psoriasis. We established that the RNA substrate is cooperatively recognized by the PIN and ZnF domains of ZC3H12C and analyzed the crystal structure of ZC3H12C bound to a single-stranded RNA substrate. The RNA engages in hydrogen-bonded contacts and stacking interactions with the PIN and ZnF domains simultaneously. The ZC3H12 ZnF shows unprecedented structural features not previously observed in any member of the CCCH-ZnF family and utilizes stacking interactions via a unique combination of spatially conserved aromatic residues to align the target transcript in a bent conformation onto the ZnF scaffold. Further comparative structural analysis of ZC3H12 CCCH-ZnF suggests that a trinucleotide sequence is recognized by ZC3H12 ZnF in target RNA. Our work not only describes the initial structure-biochemical study on ZC3H12C, but also provides the first molecular insight into RNA recognition by a ZC3H12 family member. Finally, our work points to an evolutionary code for RNA recognition adopted by CCCH-type ZnF proteins.


Assuntos
RNA/química , Ribonucleases/química , Regiões 3' não Traduzidas , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Magnésio , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Ribonucleases/metabolismo , Dedos de Zinco
15.
New Phytol ; 231(3): 1249-1264, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33932295

RESUMO

In self-incompatible Petunia species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific nonself S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S3 -RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self-pollinated and cross-pollinated pistils, indicating its occurrence before PhS3 -RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross-pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3 -RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction. Taken together, we demonstrate that PhS3 -RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.


Assuntos
Petunia , Petunia/genética , Petunia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Ubiquitinação
16.
J Phys Chem Lett ; 12(21): 5201-5207, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34038133

RESUMO

The phototriggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns time scale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 µs, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Peptídeos/metabolismo , Ribonucleases/metabolismo , Sequência de Aminoácidos , Compostos Azo/química , Compostos Azo/efeitos da radiação , Proteínas Intrinsicamente Desordenadas/química , Cinética , Luz , Peptídeos/química , Ligação Proteica/efeitos da radiação , Conformação Proteica em alfa-Hélice , Desdobramento de Proteína/efeitos da radiação , Ribonucleases/química
17.
Nucleic Acids Res ; 49(11): 6489-6510, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34038562

RESUMO

The CCR4 and CAF1 deadenylases physically interact to form the CCR4-CAF1 complex and function as the catalytic core of the larger CCR4-NOT complex. Together, they are responsible for the eventual removal of the 3'-poly(A) tail from essentially all cellular mRNAs and consequently play a central role in the posttranscriptional regulation of gene expression. The individual properties of CCR4 and CAF1, however, and their respective contributions in different organisms and cellular environments are incompletely understood. Here, we determined the crystal structure of a human CCR4-CAF1 complex and characterized its enzymatic and substrate recognition properties. The structure reveals specific molecular details affecting RNA binding and hydrolysis, and confirms the CCR4 nuclease domain to be tethered flexibly with a considerable distance between both enzyme active sites. CCR4 and CAF1 sense nucleotide identity on both sides of the 3'-terminal phosphate, efficiently differentiating between single and consecutive non-A residues. In comparison to CCR4, CAF1 emerges as a surprisingly tunable enzyme, highly sensitive to pH, magnesium and zinc ions, and possibly allowing distinct reaction geometries. Our results support a picture of CAF1 as a primordial deadenylase, which gets assisted by CCR4 for better efficiency and by the assembled NOT proteins for selective mRNA targeting and regulation.


Assuntos
Exorribonucleases/química , Proteínas Repressoras/química , Ribonucleases/química , Domínio Catalítico , Cristalografia por Raios X , Exorribonucleases/metabolismo , Fungos/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Magnésio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Zinco
18.
J Virol ; 95(13): e0033621, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33853967

RESUMO

To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol II-transcribed RNA in a ribosome-, 5' cap-, and 3' poly(A) tail-independent manner. Moreover, purified glutathione S-transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PP-induced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response.


Assuntos
Calicivirus Felino/crescimento & desenvolvimento , Evasão da Resposta Imune/imunologia , Peptídeo Hidrolases/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Animais , Infecções por Caliciviridae/patologia , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Gatos , Linhagem Celular , Células HEK293 , Humanos , Evasão da Resposta Imune/genética , Peptídeo Hidrolases/genética , Biossíntese de Proteínas/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonucleases/genética , Replicação Viral
19.
J Exp Med ; 218(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33822844

RESUMO

Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that Regnase-1 prevents severe autoimmune pathology and show its essential role in maintaining B cell homeostasis. Using Cre driver mice for ablation of Regnase-1 at various stages of B cell development, we demonstrate that loss of Regnase-1 leads to aberrant B cell activation and differentiation, resulting in systemic autoimmunity and early morbidity. The basis of these findings was informed by gene expression data revealing a regulatory role for Regnase-1 in the suppression of a transcriptional program that promotes B cell activation, survival, and differentiation. Overall, our study shows that Regnase-1 exerts critical control of B cell activation, which is required for prevention of immunopathology.


Assuntos
Autoimunidade/genética , Linfócitos B/metabolismo , Homeostase/genética , Ativação Linfocitária/genética , Ribonucleases/genética , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Camundongos Knockout , Camundongos Transgênicos , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribonucleases/metabolismo
20.
Mycoses ; 64(7): 763-770, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33797129

RESUMO

BACKGROUND: Tinea pedis is often chronic or recurrent, but not all individuals are equally susceptible to this infection. Dermatophytes are able to induce the expression of antimicrobial peptides and proteins (AMPs) in human keratinocytes and certain AMPs can inhibit the growth of dermatophytes. OBJECTIVE: The focus of this study was to analyse the secretion of relevant AMPs, especially RNase 7, human beta-defensin-2 (hBD-2) and the S-100 protein psoriasin (S100A7), in patients with confirmed tinea pedis. METHODS: To verify the diagnosis, skin scales were obtained from all patients (n = 13) and the dermatophytes were identified by potassium hydroxide mount, culture and molecular analysis. To determine the AMP concentrations, the lesional skin area of the foot was rinsed with a buffer that was subsequently analysed by ELISA. The corresponding area of the other unaffected foot as well as defined healthy skin areas of the forearm and forehead and samples from age and gender-matched healthy volunteers served as controls. RESULTS: In tinea pedis patients the AMP concentrations were higher in lesional skin than in non-lesional skin and in healthy skin of controls. In particular, concentrations of hBD-2 and psoriasin were significantly elevated. CONCLUSIONS: The induction of AMPs in tinea pedis might be triggered directly by the dermatophytes; furthermore, attendant inflammation and/or differentiation processes may play a role. Our results indicate that there is no defect in the constitutive expression and induction of the analysed AMPs by dermatophytes in the epidermis of affected patients. However, it is not known why the elevated AMP concentrations fail to efficiently combat dermatophyte growth.


Assuntos
Proteínas Citotóxicas Formadoras de Poros/metabolismo , Tinha dos Pés/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Arthrodermataceae/imunologia , Defensinas/metabolismo , Feminino , Humanos , Imunidade Inata , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Ribonucleases/metabolismo , Proteína A7 Ligante de Cálcio S100/metabolismo , Pele/metabolismo , Pele/microbiologia , Dermatopatias Infecciosas/imunologia , Dermatopatias Infecciosas/microbiologia
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