RESUMO
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. including some of the researchers involved in the 2013 work investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.
Assuntos
RNA Polimerase II , RNA Mensageiro , Fatores de Transcrição , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade de RNA , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de Zinco , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Citoplasma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.
Assuntos
Nucléolo Celular , Família Multigênica , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Animais , Humanos , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Cromatina/metabolismo , Cromatina/genética , Linhagem da Célula/genética , Dedos de Zinco/genética , Diferenciação Celular/genética , Ligação ProteicaRESUMO
BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.
Assuntos
Adenosina , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Células HCT116 , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Telômero/metabolismo , Telômero/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Enhancing drought tolerance in crops and understanding the underlying mechanisms have been subject of intense research. The precise function and molecular mechanisms of B-box zinc finger proteins (BBX) remain elusive. Here, we report a natural allele of BBX18 (BBX18TT) that encodes a C-terminal truncated protein. While most wild tomato germplasms contain the BBX18CC allele and show more drought tolerant, modern cultivated tomatoes mostly carry BBX18TT allele and are more drought sensitive. Knockout of BBX18 leads to improved drought tolerance in transgenic plants of cultivated tomato. Ascorbate peroxidase 1 (APX1) is identified as a BBX18-interacting protein that acts as a positive regulator of drought resistance in tomato. Chromatin immunoprecipitation sequencing analyses reveal that BBX18 binds to a unique cis-acting element of the APX1 promoter and represses its gene expression. This study provides insights into the molecular mechanism underlying drought resistance mediated by the BBX18-APX1 module in plants.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Solanum lycopersicum , Fatores de Transcrição , Dedos de Zinco , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Dedos de Zinco/genética , Regiões Promotoras Genéticas/genética , Ascorbato Peroxidases/metabolismo , Ascorbato Peroxidases/genética , AlelosRESUMO
The zinc-finger antiviral protein (ZAP) is a restriction factor that proficiently impedes the replication of a variety of RNA and DNA viruses. In recent years, the affinity of ZAP's zinc-fingers for single-stranded RNA (ssRNA) rich in CpG dinucleotides was uncovered. High frequencies of CpGs in RNA may suggest a non-self origin, which underscores the importance of ZAP as a potential cellular sensor of (viral) RNA. Upon binding viral RNA, ZAP recruits cellular cofactors to orchestrate a finely tuned antiviral response that limits virus replication via distinct mechanisms. These include promoting degradation of viral RNA, inhibiting RNA translation, and synergizing with other immune pathways. Depending on the viral species and experimental set-up, different isoforms and cellular cofactors have been reported to be dominant in shaping the ZAP-mediated antiviral response. Here we review how ZAP differentially affects viral replication depending on distinct interactions with RNA, cellular cofactors, and viral proteins to discuss how these interactions shape the antiviral mechanisms that have thus far been reported for ZAP. Importantly, we zoom in on the unknown aspects of ZAP's antiviral system and its therapeutic potential to be employed in vaccine design.
Assuntos
Proteínas de Ligação a RNA , Viroses , Replicação Viral , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Viroses/metabolismo , Viroses/imunologia , RNA Viral/metabolismo , Animais , Dedos de ZincoRESUMO
The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.
Assuntos
Hemólise , Fatores de Transcrição Kruppel-Like , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Animais , Camundongos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Mutação de Sentido Incorreto , Humanos , Anemia Hemolítica/genética , Anemia Hemolítica/metabolismo , Camundongos Knockout , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Masculino , Dedos de Zinco , Feminino , MutaçãoRESUMO
Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Nucleossomos , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Microscopia Crioeletrônica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Ilhas de CpG , Ubiquitinação , Evolução Molecular , DNA/metabolismo , DNA/química , DNA/genética , Dedos de Zinco , Cromatina/metabolismo , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Eucariotos/genética , Eucariotos/metabolismo , Ligação Proteica , Histonas/metabolismo , Histonas/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/químicaRESUMO
KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.
Assuntos
Catharanthus , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Oxilipinas , Proteínas de Plantas , Fatores de Transcrição , Catharanthus/genética , Catharanthus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Dedos de Zinco CYS2-HIS2/genética , Plantas Geneticamente Modificadas , Alcaloides de Triptamina e Secologanina/metabolismo , Filogenia , Dedos de ZincoRESUMO
CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas , Rosa , Estresse Fisiológico , Rosa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Genoma de Planta , Regiões Promotoras Genéticas , Estudo de Associação Genômica Ampla , Perfilação da Expressão Gênica , Dedos de Zinco/genéticaRESUMO
The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases , Metilação de DNA , Dedos de Zinco , Humanos , Animais , Camundongos , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/química , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/metabolismo , Ilhas de CpG/genética , DNA/metabolismo , DNA/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Mutação , Ligação Proteica , Nucleossomos/metabolismo , Nucleossomos/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Domínios Proteicos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Face/anormalidades , Proteínas NuclearesRESUMO
Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ligação a DNA , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neurônios Motores/metabolismo , Diferenciação Celular , Dedos de Zinco , Humanos , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
Protein methylation is a functionally important post-translational modification that occurs on diverse amino acid residues. The current proteomics approaches are inefficient to discover the methylation on residues other than Arg and Lys, which hinders the deep understanding of the functional role of rare protein methylation. Herein, we present a methyl-specific metabolic labeling approach for global methylome mapping, which enable the acquisition of methylome dataset covering diverse methylation types. Interestingly, of the identified methylation events, His methylation is found to be preferably occurred in C3H1 zinc fingers (ZFs). These His methylation events are determined to be Nπ specific and catalyzed by CARNMT1. The His methylation is found to stabilize the structure of ZFs. U2AF1 is used as a proof-of-concept to highlight the functional importance of His methylation in ZFs in RNA binding and RNA metabolism. The results of this study enable novel understanding of how protein methylation regulates cellular processes.
Assuntos
Histidina , Processamento de Proteína Pós-Traducional , Dedos de Zinco , Histidina/metabolismo , Metilação , Humanos , Epigenoma , Células HEK293 , Metiltransferases/metabolismo , Metiltransferases/genéticaRESUMO
Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif 'QALGGH' were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.
Assuntos
Dedos de Zinco CYS2-HIS2 , Regulação da Expressão Gênica de Plantas , Filogenia , Pinus , Proteínas de Plantas , Estresse Fisiológico , Fatores de Transcrição , Pinus/genética , Pinus/parasitologia , Pinus/metabolismo , Estresse Fisiológico/genética , Dedos de Zinco CYS2-HIS2/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Dedos de ZincoRESUMO
Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal-ligand H23 found in odd-numbered zinc fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711, it has been thought that Z7 is probably non-functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal-ligand. The Z7 domain adopts a stable tertiary structure upon metal-binding. The NMR structure of Zn2+-bound Z7 shows the classical ßßα-fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal-binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring-flipping of phenylalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the Kd values for Zn2+ and Co2+ and retain the high thermal stability of the WT domain above 80°C. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4-nitrophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Fatores de Transcrição , Dedos de Zinco , Humanos , Sítios de Ligação , Modelos Moleculares , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zinco/metabolismo , Zinco/químicaRESUMO
Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.
Assuntos
Proteínas de Ligação a DNA , DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA/metabolismo , DNA/química , DNA/genética , Humanos , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Dedos de Zinco , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/química , Sequências de Repetição em Tandem , Sequência de Aminoácidos , Bases de Dados de Proteínas , Sítios de Ligação/genéticaRESUMO
Human ZC3H11A is an RNA-binding zinc finger protein involved in mRNA export and required for the efficient growth of human nuclear replicating viruses. Its biochemical properties are largely unknown so our goal has been to produce the protein in a pure and stable form suitable for its characterization. This has been challenging since the protein is large (810 amino acids) and with only the N-terminal zinc finger domain (amino acids 1-86) being well structured, the remainder is intrinsically disordered. Our production strategies have encompassed recombinant expression of full-length, truncated and mutated ZC3H11A variants with varying purification tags and fusion proteins in several expression systems, with or without co-expression of chaperones and putative interaction partners. A range of purification schemes have been explored. Initially, only truncated ZC3H11A encompassing the zinc finger domain could successfully be produced in a stable form. It required recombinant expression in insect cells since expression in E. coli gave a protein that aggregated. To reduce problematic nucleic acid contaminations, Cys8, located in one of the zinc fingers, was substituted by Ala and Ser. Interestingly, this did not affect nucleic acid binding, but the full-length protein was stabilised while the truncated version was insoluble. Ultimately, we discovered that when using alkaline buffers (pH 9) for purification, full-length ZC3H11A expressed in Sf9 insect cells was obtained in a stable and >90 % pure form, and as a mixture of monomers, dimers, tetramers and hexamers. Many of the challenges experienced are consistent with its predicted structure and unusual charge distribution.
Assuntos
Escherichia coli , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Animais , Dedos de Zinco , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Células Sf9 , Estabilidade Proteica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/biossínteseRESUMO
Small ubiquitin-binding domains (UBDs) recognize small surface patches on ubiquitin with weak affinity, and it remains a conundrum how specific cellular responses may be achieved. Npl4-type zinc-finger (NZF) domains are â¼30 amino acid, compact UBDs that can provide two ubiquitin-binding interfaces, imposing linkage specificity to explain signaling outcomes. We here comprehensively characterize the linkage preference of human NZF domains. TAB2 prefers Lys6 and Lys63 linkages phosphorylated on Ser65, explaining why TAB2 recognizes depolarized mitochondria. Surprisingly, most NZF domains do not display chain linkage preference, despite conserved, secondary interaction surfaces. This suggests that some NZF domains may specifically bind ubiquitinated substrates by simultaneously recognizing substrate and an attached ubiquitin. We show biochemically and structurally that the NZF1 domain of the E3 ligase HOIPbinds preferentially to site-specifically ubiquitinated forms of NEMO and optineurin. Thus, despite their small size, UBDs may impose signaling specificity via multivalent interactions with ubiquitinated substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ligação Proteica , Ubiquitina , Humanos , Especificidade por Substrato , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Dedos de Zinco , Ubiquitinação , Quinase I-kappa B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Domínios Proteicos , Fosforilação , Células HEK293 , Proteínas de Membrana TransportadorasRESUMO
Rhizome rot is a destructive soil-borne disease of Polygonatum kingianum and adversely affects the yield and sustenance of the plant. Understanding how the causal fungus Fusarium oxysporum infects P. kingianum may suggest effective control measures against rhizome rot. In germinating conidia of infectious F. oxysporum, expression of the zinc finger transcription factor gene Zfp1, consisting of two C2H2 motifs, was up-regulated. To characterize the critical role of ZFP1, we generated independent deletion mutants (zfp1) and complemented one mutant with a transgenic copy of ZFP1 (zfp1 tZFP1). Mycelial growth and conidial production of zfp1 were slower than those of wild type (ZFP1) and zfp1 tZFP1. Additionally, a reduced inhibition of growth suggested zfp1 was less sensitive to conditions promoting cell wall and osmotic stresses than ZFP1 and zfp1 tZFP1. Furthermore pathogenicity tests suggested a critical role for growth of zfp1 in infected leaves and rhizomes of P. kingianum. Thus ZFP1 is important for mycelial growth, conidiation, osmoregulation, and pathogenicity in P. kingianum.
Assuntos
Proteínas Fúngicas , Fusarium , Osmorregulação , Doenças das Plantas , Polygonatum , Esporos Fúngicos , Fatores de Transcrição , Dedos de Zinco , Fusarium/patogenicidade , Fusarium/genética , Fusarium/crescimento & desenvolvimento , Fusarium/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/genética , Virulência/genética , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Polygonatum/microbiologia , Regulação Fúngica da Expressão GênicaRESUMO
Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3'-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich RNAs. We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence features through the ROQ-ZnF tandem.
Assuntos
Ligação Proteica , Proteínas de Ligação a RNA , Dedos de Zinco , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Humanos , Modelos Moleculares , RNA/química , RNA/metabolismo , RNA/genética , Sítios de Ligação , Regiões 3' não Traduzidas , RNA Mensageiro/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Estabilidade de RNA , Ubiquitina-Proteína LigasesRESUMO
To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.