RESUMO
TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51-like kinase complex during autophagosome formation. Through tank-binding kinase 1-mediated phosphorylation of the TNIP1 FIP200 interacting region (FIR) motif, the binding affinity of TNIP1 for FIP200, a component of the Unc-51-like kinase complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIR motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy.
RESUMO
Eukaryotes contain two sets of genomes: the nuclear genome and the mitochondrial genome. The mitochondrial genome transcripts 13 mRNAs that encode 13 essential proteins for the oxidative phosphorylation complex, 2 rRNAs (12s rRNA and 16s rRNA), and 22 tRNAs. The proper assembly and maturation of the mitochondrial ribosome (mitoribosome) are critical for the translation of the 13 key proteins and the function of the mitochondrion. Human ribosome-binding factor A (hsRBFA) is a mitoribosome assembly factor that binds with helix 28, helix 44 and helix 45 of 12S rRNA and facilitates the transcriptional modification of 12S rRNA during the mitoribosomal biogenesis. Previous research mentioned that the malfunction of hsRBFA will induce the instability of mitoribosomes and affect the function of mitochondria, but the mechanisms underlying the interaction between hsRBFA and 12S rRNA and its influence on mitochondrial function are still unknown. In this study, we found that hsRBFA binds with double strain RNA (dsRNA) through its whole N-terminus (Nt) instead of the KH-like domain alone, which is different from the other homologous. Furthermore, we mapped the key residues that affected the RNA binding and maturation of mitoribosomes in vitro. Finally, we investigated how these residues affect mitochondrial functions in detail and systematically.
Assuntos
Proteínas Mitocondriais , Ribossomos Mitocondriais , RNA Ribossômico , Proteínas de Ligação a RNA , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas Ribossômicas/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Multiple proteins bind to telomeric DNA and are important for the role of telomeres in genome stability. A recent study established a broad-complex, tramtrack and bric-à-brac - zinc finger (BTB-ZF) protein, ZBTB10 (zinc finger and BTB domain-containing protein 10), as a telomeric variant repeat-binding protein at telomeres that use an alternative method for lengthening telomeres). ZBTB10 specifically interacts with the double-stranded telomeric variant repeat sequence TTGGGG by employing its tandem C2H2 zinc fingers (ZF1-2). Here, we solved the crystal structure of human ZBTB10 ZF1-2 in complex with a double-stranded DNA duplex containing the sequence TTGGGG to assess the molecular details of this interaction. Combined with calorimetric analysis, we identified the vital residues in TTGGGG recognition and determined the specific recognition mechanisms that are different from those of TZAP (telomere zinc finger-associated protein), a recently defined telomeric DNA-binding protein. Following these studies, we further identified a single amino-acid mutant (Arg767Gln) of ZBTB10 ZF1-2 that shows a preference for the telomeric DNA repeat TTAGGG sequence. We solved the cocrystal structure, providing a structural basis for telomeric DNA recognition by C2H2 ZF proteins.
Assuntos
Proteínas de Ligação a DNA , Proteínas Repressoras , Humanos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Telômero/metabolismo , Dedos de Zinco/genéticaRESUMO
The molecular mechanisms of aging are unsolved fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding with the 3' untranslated regions (3' UTR) in the target mRNAs. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF-1, a mitochondrial fission factor, and subsequently regulated longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. Those mutations reduced the fertility and extended the lifespan of C. elegans. Deep sequencing of total mRNAs from wild-type and puf-8 mutant worms as well as in vivo RNA Crosslinking and Immunoprecipitation (CLIP) experiments identified six PUF-8 regulated genes, which contain at least one PUF-binding element (PBE) at the 3' UTR. One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8 mutant animals. We conclude that PUF-8 regulate the lifespan of C. elegans may not only via MFF but also via modulating pqm-1-related pathways.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Longevidade/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Envelhecimento/genética , Animais , Cristalografia por Raios X , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Membrana/metabolismo , Dinâmica Mitocondrial/genética , Mitofagia/genética , Modelos Animais , Conformação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transativadores/genéticaRESUMO
The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate 'cluster assistance' in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Células HCT116 , Células HEK293 , Humanos , Células K562 , MicroRNAs/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Liquid-liquid phase separation (LLPS) of biomolecules drives the formation of subcellular compartments with distinct physicochemical properties. These compartments, free of lipid bilayers and therefore called membraneless organelles, include nucleoli, centrosomes, heterochromatin, and centromeres. These have emerged as a new paradigm to account for subcellular organization and cell fate decisions. Here we summarize recent studies linking LLPS to mitotic spindle, heterochromatin, and centromere assembly and their plasticity controls in the context of the cell division cycle, highlighting a functional role for phase behavior and material properties of proteins assembled onto heterochromatin, centromeres, and central spindles via LLPS. The techniques and tools for visualizing and harnessing membraneless organelle dynamics and plasticity in mitosis are also discussed, as is the potential for these discoveries to promote new research directions for investigating chromosome dynamics, plasticity, and interchromosome interactions in the decision-making process during mitosis.
Assuntos
Tomada de Decisões , Extração Líquido-Líquido , Divisão Celular , Humanos , Mitose , Organelas/metabolismoRESUMO
A protein-RNA complex containing the RNA helicase CGH-1 and a germline specific RNA-binding protein CAR-1 is involved in various aspects of function in C. elegans. However, the structural basis for the assembly of this protein complex remains unclear. Here, we elucidate the molecular basis of the recognition of CGH-1 by CAR-1. Additionally, we found that the ATPase activity of CGH-1 is stimulated by NTL-1a MIF4G domain in vitro. Furthermore, we determined the structures of the two RecA-like domains of CGH-1 by X-ray crystallography at resolutions of 1.85 and 2.40 Å, respectively. Structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1. NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR-1, assessed by ITC and GST-pulldown in vitro. These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1. Together, our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Aminoácidos/química , Animais , Sequência Conservada , Cristalografia por Raios X , Isótopos de Nitrogênio , Domínios Proteicos , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Terminal uridylyl transferase (TUTase) is one type of enzyme that modifies RNA molecules by facilitating the post-transcriptional addition of uridyl ribonucleotides to their 3' ends. Recent researches have reported that Drosophila TUTase, Tailor, exhibits an intrinsic preference for RNA substrates ending in 3'G, distinguishing it from any other known TUTases. Through this unique feature, Tailor plays a crucial role as the repressor in the biogenesis pathway of splicing-derived mirtron pre-miRNAs. Here we describe crystal structures of core catalytic domain of Tailor and its complexes with RNA stretches 5'-AGU-3' and 5'-AGUU-3'. We demonstrate that R327 and N347 are two key residues contributing cooperatively to Tailor's preference for 3'G, and R327 may play an extra role in facilitating the extension of polyuridylation chain. We also demonstrate that conformational stability of the exit of RNA-binding groove also contributes significantly to Tailor's activity. Overall, our work reveals useful insights to explain why Drosophila Tailor can preferentially select RNA substrates ending in 3'G and provides important values for further understanding the biological significances of biogenesis pathway of mirtron in flies.
Assuntos
Proteínas de Drosophila/genética , Drosophila/enzimologia , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/química , RNA/biossíntese , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico/genética , Drosophila/genética , Proteínas de Drosophila/química , Guanina/química , MicroRNAs/genética , Nucleotidiltransferases/química , RNA/genética , RNA Nucleotidiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , Especificidade por SubstratoRESUMO
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). Here, we report a 2.90Å crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, our research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Assuntos
Proteínas Cromossômicas não Histona/química , DNA Helicases/química , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/química , RNA de Cadeia Dupla/genética , Fatores de Transcrição/química , Animais , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , RNA de Cadeia Dupla/química , Fatores de Transcrição/genética , Cromossomo X/genéticaRESUMO
Telomeres are nucleoprotein structures at the ends of linear chromosomes and present an essential feature for genome integrity. Vertebrate telomeres usually consist of hexameric TTAGGG repeats, however, in cells that use the alternative lengthening of telomeres (ALT) mechanism, variant repeat sequences are interspersed throughout telomeres. Previously, it was shown that NR2C/F transcription factors bind to TCAGGG variant repeats and contribute to telomere maintenance in ALT cells. While specific binders to other variant repeat sequences have been lacking to date, we here identify ZBTB10 as the first TTGGGG-binding protein and demonstrate direct binding via the two zinc fingers with affinity in the nanomolar range. Concomitantly, ZBTB10 co-localizes with a subset of telomeres in ALT-positive U2OS cells and interacts with TRF2/RAP1 via the N-terminal region of TRF2. Our data establishes ZBTB10 as a novel variant repeat binding protein at ALT telomeres.
Assuntos
Proteínas Repressoras/genética , Homeostase do Telômero/genética , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Sítios de Ligação/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Humanos , Ligação Proteica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/genéticaRESUMO
Mitochondria are essential molecular machinery for the maintenance of cellular energy supply by the oxidative phosphorylation system (OXPHOS). Mitochondrial transcription factor B1 (TFB1M) is a dimethyltransferase that maintains mitochondrial homeostasis by catalyzing dimethylation of two adjacent adenines located in helix45 (h45) of 12S rRNA. This m62A modification is indispensable for the assembly and maturation of human mitochondrial ribosomes. However, both the mechanism of TFB1M catalysis and the precise function of TFB1M in mitochondrial homeostasis are unknown. Here we report the crystal structures of a ternary complex of human (hs) TFB1M-h45-S-adenosyl-methionine and a binary complex hsTFB1M-h45. The structures revealed a distinct mode of hsTFB1M interaction with its rRNA substrate and with the initial enzymatic state involved in m62A modification. The suppression of hsTFB1M protein level or the overexpression of inactive hsTFB1M mutants resulted in decreased ATP production and reduced expression of components of the mitochondrial OXPHOS without affecting transcription of the corresponding genes and their localization to the mitochondria. Therefore, hsTFB1M regulated the translation of mitochondrial genes rather than their transcription via m62A modification in h45.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Mitocondriais/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA Ribossômico/genética , Fatores de Transcrição/genética , Sequência de Bases , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Homeostase/genética , Humanos , Metilação , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Mutação , Fosforilação Oxidativa , Ligação Proteica , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
3' uridylation is an essential modification associated with coding and noncoding RNA degradation in eukaryotes. In Arabidopsis, HESO1 was first identified as the major nucleotidyl transferase that uridylates most unmethylated miRNAs, and URT1 was later reported to play a redundant but important role in miRNA uridylation when HESO1 is absent. Two enzymes work sequentially and collaboratively to tail different forms of the same miRNAs in vivo. For mRNA, however, URT1 becomes the main enzyme to uridylate the majority of mRNA and repairs their deadenylated ends to restore the binding site for Poly(A) Binding Protein (PABP). HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails in fulfilling the same task. To understand the structural basis these two functional homologues possess for their different substrate preferences and catalytic behaviors, we first determined the crystal structures of URT1 in the absence and presence of UTP. Our structures, together with functional assay and sequence analysis, indicated that URT1 has a conserved UTP-recognition mechanism analogue to the terminal uridylyl transferases from other species whereas HESO1 may evolve separately to recognize UTP in a different way. Moreover, URT1 N552 may be an important residue in interacting with 3' nucleotide of RNA substrate. The URT1 structure we determined represents the first structure of uridylyl transferase from plants, shedding light on the mechanisms of URT1/HESO1-dependent RNA metabolism.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , RNA Nucleotidiltransferases/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RNA Nucleotidiltransferases/metabolismo , Uridina Trifosfato/metabolismoRESUMO
Methyltransferase RlmCD was previously shown to be responsible for the introduction of C5 methylation at both U747 and U1939 of the 23S ribosomal RNA in Streptococcus pneumoniae. Intriguingly, its structural homologue, RumA, can only catalyze the methylation of U1939, while RlmC is the dedicated enzyme for m5U747 in Escherichia coli. In this study, we describe the structure of RlmCD in complex with its cofactor and the RNA substrate containing U747 at 2.00 Å or U1939 at 3.10 Å. We demonstrate that multiple structural features collaborate to establish the dual enzymatic activities of RlmCD. Of them, the side-chain rearrangement of F145 was observed to be an unusual mechanism through which RlmCD can discriminate between U747- and U1939-containing RNA substrate by switching the intermolecular aromatic stacking between protein and RNA on/off. An in-vitro methyltransferase assay and electrophoretic mobility shift assay were performed to validate these findings. Overall, our complex structures allow for a better understanding of the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of the RumA-type enzyme and the potential development of antibiotic drugs against S. pneumoniae.
Assuntos
Metiltransferases/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Escherichia coli/metabolismo , Metiltransferases/genética , Elementos Estruturais de Proteínas , Estrutura Terciária de Proteína/genética , RNA/metabolismo , RNA Ribossômico 23S/metabolismo , Streptococcus pneumoniae/genética , Relação Estrutura-AtividadeRESUMO
Histone acetylation is a hallmark for gene transcription. As a histone acetyltransferase, MOZ (monocytic leukemia zinc finger protein) is important for HOX gene expression as well as embryo and postnatal development. In vivo, MOZ forms a tetrameric complex with other subunits, including several chromatin-binding modules with regulatory functions. Here we report the solution structure of the tandem PHD (plant homeodomain) finger (PHD12) of human MOZ in a free state and the 1.47 Å crystal structure in complex with H3K14ac peptide, which reveals the structural basis for the recognition of unmodified R2 and acetylated K14 on histone H3. Moreover, the results of chromatin immunoprecipitation (ChIP) and RT-PCR assays indicate that PHD12 facilitates the localization of MOZ onto the promoter locus of the HOXA9 gene, thereby promoting the H3 acetylation around the promoter region and further up-regulating the HOXA9 mRNA level. Taken together, our findings suggest that the combinatorial readout of the H3R2/K14ac by PHD12 might represent an important epigenetic regulatory mechanism that governs transcription and also provide a clue of cross-talk between the MOZ complex and histone H3 modifications.
Assuntos
Arginina/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Lisina/metabolismo , Transcrição Gênica , Acetilação , Sequência de Aminoácidos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD-ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.
Assuntos
Proteínas de Ligação a DNA , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Proteína 4 de Ligação ao Retinoblastoma , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/química , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Relação Estrutura-Atividade , Telômero/química , Telômero/genética , Telômero/metabolismoRESUMO
In bacteria, small non-coding RNAs (sRNAs) could function in gene regulations under variable stress responses. DsrA is an â¼90-nucleotide Hfq-dependent sRNA found in Escherichia coli. It regulates the translation and degradation of multiple mRNAs, such as rpoS, hns, mreB and rbsD mRNAs. However, its functional structure and particularly how it regulates multiple mRNAs remain obscure. Using NMR, we investigated the solution structures of the full-length and isolated stem-loops of DsrA. We first solved the NMR structure of the first stem-loop (SL1), and further studied the melting process of the SL1 induced by the base-pairing with the rpoS mRNA and the A-form duplex formation of the DsrA/rpoS complex. The secondary structure of the second stem-loop (SL2) was also determined, which contains a lower stem and an upper stem with distinctive stability. Interestingly, two conformational states of SL2 in dynamic equilibrium were observed in our NMR spectra, suggesting that the conformational selection may occur during the base-pairing between DsrA and mRNAs. In summary, our study suggests that the conformational plasticity of DsrA may represent a special mechanism sRNA employed to deal with its multiple regulatory targets of mRNA.
Assuntos
Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Proteínas de Bactérias/genética , Pareamento de Bases , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Fator sigma/genéticaRESUMO
PUF (Pumilio/fem-3 mRNA binding factor) proteins, a conserved family of RNA-binding proteins, recognize specific single-strand RNA targets in a specific modular way. Although plants have a greater number of PUF protein members than do animal and fungal systems, they have been the subject of fewer structural and functional investigations. The aim of this study was to elucidate the involvement of APUM23, a nucleolar PUF protein in the plant Arabidopsis, in pre-rRNA processing. APUM23 is distinct from classical PUF family proteins, which are located in the cytoplasm and bind to 3'UTRs of mRNA to modulate mRNA expression and localization. We found that the complete RNA target sequence of APUM23 comprises 11 nt in 18S rRNA at positions 1141-1151. The complex structure shows that APUM23 has 10 PUF repeats; it assembles into a C-shape, with an insertion located within the inner concave surface. We found several different RNA recognition features. A notable structural feature of APUM23 is an insertion in the third PUF repeat that participates in nucleotide recognition and maintains the correct conformation of the target RNA. Our findings elucidate the mechanism for APUM23's-specific recognition of 18S rRNA.
Assuntos
Proteínas de Arabidopsis/metabolismo , RNA de Plantas/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Sítios de Ligação/genética , Calorimetria/métodos , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , RNA de Plantas/química , RNA de Plantas/genética , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , TermodinâmicaRESUMO
Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA and target mRNA. Hfq mediates DsrA-dependent translational activation of rpoS mRNA at low temperatures. rpoS encodes the stationary-phase σ factor σ(S), which is the central regulator in general stress response. However, structural information on Hfq-DsrA interaction is not yet available. Although Hfq is reported to hydrolyze ATP, the ATP-binding site is still unknown. Here, we report a ternary crystal complex structure of Escherichia coli Hfq bound to a major Hfq recognition region on DsrA (AU(6)A) together with ADP, and a crystal complex structure of Hfq bound to ADP. AU(6)A binds to the proximal and distal sides of two Hfq hexamers. ADP binds to a purine-selective site on the distal side and contacts conserved arginine or glutamine residues on the proximal side of another hexamer. This binding mode is different from previously postulated. The cooperation of two different Hfq hexamers upon nucleic acid binding in solution is verified by fluorescence polarization and solution nuclear magnetic resonance (NMR) experiments using fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with full-length Hfq and DsrA also supports cooperation of Hfq hexamers upon DsrA binding. The implications of Hfq hexamer cooperation have been discussed.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/química , Modelos Moleculares , Pequeno RNA não Traduzido/metabolismo , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Polímeros , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
In higher eukaryotes, the centromere is epigenetically specified by the histone H3 variant Centromere Protein-A (CENP-A). Deposition of CENP-A to the centromere requires histone chaperone HJURP (Holliday junction recognition protein). The crystal structure of an HJURP-CENP-A-histone H4 complex shows that HJURP binds a CENP-A-H4 heterodimer. The C-terminal ß-sheet domain of HJURP caps the DNA-binding region of the histone heterodimer, preventing it from spontaneous association with DNA. Our analysis also revealed a novel site in CENP-A that distinguishes it from histone H3 in its ability to bind HJURP. These findings provide key information for specific recognition of CENP-A and mechanistic insights into the process of centromeric chromatin assembly.
Assuntos
Autoantígenos/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Histonas/química , Modelos Moleculares , Autoantígenos/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.