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1.
Nucleic Acids Res ; 48(17): 9637-9648, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890394

RESUMO

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3-AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3-AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3-AG tetramer. SEP3 and SEP3-AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3-AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3-AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.


Assuntos
Proteína AGAMOUS de Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodomínio/metabolismo , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Proteína AGAMOUS de Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Proteínas de Transporte/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Proteínas de Homeodomínio/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Fatores de Transcrição/genética
2.
Proc Natl Acad Sci U S A ; 117(38): 23982-23990, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32887800

RESUMO

MAC5 is a component of the conserved MOS4-associated complex. It plays critical roles in development and immunity. Here we report that MAC5 is required for microRNA (miRNA) biogenesis. MAC5 interacts with Serrate (SE), which is a core component of the microprocessor that processes primary miRNA transcripts (pri-miRNAs) into miRNAs and binds the stem-loop region of pri-miRNAs. MAC5 is essential for both the efficient processing and the stability of pri-miRNAs. Interestingly, the reduction of pri-miRNA levels in mac5 is partially caused by XRN2/XRN3, the nuclear-localized 5'-to-3' exoribonucleases, and depends on SE. These results reveal that MAC5 plays a dual role in promoting pri-miRNA processing and stability through its interaction with SE and/or pri-miRNAs. This study also uncovers that pri-miRNAs need to be protected from nuclear RNA decay machinery, which is connected to the microprocessor.


Assuntos
Proteínas de Arabidopsis , Exorribonucleases , MicroRNAs , Proteínas Nucleares , Proteínas de Ligação a RNA , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
3.
Mol Pharmacol ; 98(4): 303-313, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32873746

RESUMO

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABAA receptor. The binding of [3H]muscimol has been used to evaluate the distribution of GABAA receptors in the brain, and studies of modulation of [3H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABAA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α1ß3 GABAA receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [3H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [3H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.


Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Muscimol/farmacologia , Receptores de GABA-A/metabolismo , Esteroides/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Células HEK293 , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Muscimol/química , Pregnanolona/farmacologia , Pregnenolona/farmacologia , Receptores de GABA-A/química , Receptores de GABA-A/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trítio/química
4.
Nucleic Acids Res ; 48(18): 10280-10296, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32955564

RESUMO

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.


Assuntos
Arginina/análogos & derivados , Fator de Iniciação 2 em Eucariotos/genética , RNA Mensageiro/genética , Substituição de Aminoácidos/genética , Arginina/genética , Códon de Iniciação/genética , Humanos , Complexos Multiproteicos/genética , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética
5.
PLoS Genet ; 16(8): e1008955, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776921

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by excess lipid accumulation in the liver without significant consumption of alcohol. The transmembrane 6 superfamily member 2 (TM6SF2) E167K missense variant strongly associates with NAFLD in humans. The E167K mutation destabilizes TM6SF2, resulting in hepatic lipid accumulation and low serum lipid levels. However, the molecular mechanism by which TM6SF2 regulates lipid metabolism remains unclear. By using tandem affinity purification in combination with mass spectrometry, we found that apolipoprotein B (APOB), ER lipid raft protein (ERLIN) 1 and 2 were TM6SF2-interacting proteins. ERLINs and TM6SF2 mutually bound and stabilized each other. TM6SF2 bound and stabilized APOB via two luminal loops. ERLINs did not interact with APOB directly but still increased APOB stability through stabilizing TM6SF2. This APOB stabilization was hampered by the E167K mutation that reduced the protein expression of TM6SF2. In mice, knockout of Tm6sf2 and knockdown of Tm6sf2 or Erlins decreased hepatic APOB protein level, causing lipid accumulation in the liver and lowering lipid levels in the serum. We conclude that defective APOB stabilization, as a result of ERLINs or TM6SF2 deficiency or E167K mutation, is a key factor contributing to NAFLD.


Assuntos
Apolipoproteína B-100/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Colesterol/genética , Colesterol/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Imunoprecipitação , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Lipídeos/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/genética , Transfecção
6.
Nucleic Acids Res ; 48(16): 9301-9319, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813020

RESUMO

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.


Assuntos
Complexos Multiproteicos/genética , Mapas de Interação de Proteínas/genética , Pequeno RNA não Traduzido/genética , RNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Fator Proteico 1 do Hospedeiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Ribossomos/genética
7.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
8.
Nucleic Acids Res ; 48(15): 8529-8544, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738045

RESUMO

Myocyte enhancer factor-2B (MEF2B) has the unique capability of binding to its DNA target sites with a degenerate motif, while still functioning as a gene-specific transcriptional regulator. Identifying its DNA targets is crucial given regulatory roles exerted by members of the MEF2 family and MEF2B's involvement in B-cell lymphoma. Analyzing structural data and SELEX-seq experimental results, we deduced the DNA sequence and shape determinants of MEF2B target sites on a high-throughput basis in vitro for wild-type and mutant proteins. Quantitative modeling of MEF2B binding affinities and computational simulations exposed the DNA readout mechanisms of MEF2B. The resulting binding signature of MEF2B revealed distinct intricacies of DNA recognition compared to other transcription factors. MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif, where A-tract polarity dictates nuances of binding. The predominant role of shape readout at the center of the core motif, with most contacts formed in the minor groove, differs from previously observed protein-DNA readout modes. MEF2B, therefore, represents a unique protein for studies of the role of DNA shape in achieving binding specificity. MEF2B-DNA recognition mechanisms are likely representative for other members of the MEF2 family.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/ultraestrutura , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/ultraestrutura , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica/genética
9.
Nucleic Acids Res ; 48(18): 10329-10341, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663306

RESUMO

The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ∼1.5-2נstronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.


Assuntos
Adenina/química , Complexos Multiproteicos/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Fatores de Processamento de RNA/química , DNA/química , DNA/genética , DNA/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Histona Desmetilases/genética , Humanos , Metilação , Metiltransferases/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/ultraestrutura , Domínios Proteicos/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/ultraestrutura , Proteínas de Ligação a RNA/genética
10.
Nat Med ; 26(8): 1264-1270, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661391

RESUMO

Cancer cachexia is a highly prevalent condition associated with poor quality of life and reduced survival1. Tumor-induced perturbations in the endocrine, immune and nervous systems drive anorexia and catabolic changes in adipose tissue and skeletal muscle, hallmarks of cancer cachexia2-4. However, the molecular mechanisms driving cachexia remain poorly defined, and there are currently no approved drugs for the condition. Elevation in circulating growth differentiation factor 15 (GDF15) correlates with cachexia and reduced survival in patients with cancer5-8, and a GDNF family receptor alpha like (GFRAL)-Ret proto-oncogene (RET) signaling complex in brainstem neurons that mediates GDF15-induced weight loss in mice has recently been described9-12. Here we report a therapeutic antagonistic monoclonal antibody, 3P10, that targets GFRAL and inhibits RET signaling by preventing the GDF15-driven interaction of RET with GFRAL on the cell surface. Treatment with 3P10 reverses excessive lipid oxidation in tumor-bearing mice and prevents cancer cachexia, even under calorie-restricted conditions. Mechanistically, activation of the GFRAL-RET pathway induces expression of genes involved in lipid metabolism in adipose tissues, and both peripheral chemical sympathectomy and loss of adipose triglyceride lipase protect mice from GDF15-induced weight loss. These data uncover a peripheral sympathetic axis by which GDF15 elicits a lipolytic response in adipose tissue independently of anorexia, leading to reduced adipose and muscle mass and function in tumor-bearing mice.


Assuntos
Caquexia/tratamento farmacológico , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Complexos Multiproteicos/ultraestrutura , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ret/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Anticorpos Monoclonais , Caquexia/complicações , Caquexia/genética , Caquexia/imunologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/ultraestrutura , Fator 15 de Diferenciação de Crescimento/ultraestrutura , Xenoenxertos , Humanos , Peroxidação de Lipídeos , Camundongos , Complexos Multiproteicos/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Neoplasias/complicações , Neoplasias/genética , Neoplasias/imunologia , Proteínas Proto-Oncogênicas c-ret/ultraestrutura , Transdução de Sinais , Perda de Peso
11.
Nucleic Acids Res ; 48(15): 8740-8754, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644155

RESUMO

In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, disuccinimidyl sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA's to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.


Assuntos
Aminoacil-tRNA Sintetases/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Conformação Proteica , Aminoacil-tRNA Sintetases/genética , Células HEK293 , Humanos , Espectrometria de Massas , Conformação Molecular , Simulação de Acoplamento Molecular , Complexos Multiproteicos/genética , Ligação Proteica
12.
Nucleic Acids Res ; 48(15): 8675-8685, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687167

RESUMO

Along with nucleobase pairing, base-base stacking interactions are one of the two main types of strong non-covalent interactions that define the unique secondary and tertiary structure of RNA. In this paper we studied two subfamilies of nucleobase-inserted stacking structures: (i) with any base intercalated between neighboring nucleotide residues (base-intercalated element, BIE, i + 1); (ii) with any base wedged into a hydrophobic cavity formed by heterocyclic bases of two nucleotides which are one nucleotide apart in sequence (base-wedged element, BWE, i + 2). We have exploited the growing database of natively folded RNA structures in Protein Data Bank to analyze the distribution and structural role of these motifs in RNA. We found that these structural elements initially found in yeast tRNAPhe are quite widespread among the tertiary structures of various RNAs. These motifs perform diverse roles in RNA 3D structure formation and its maintenance. They contribute to the folding of RNA bulges and loops and participate in long-range interactions of single-stranded stretches within RNA macromolecules. Furthermore, both base-intercalated and base-wedged motifs participate directly or indirectly in the formation of RNA functional centers, which interact with various ligands, antibiotics and proteins.


Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/ultraestrutura , RNA/ultraestrutura , Antibacterianos/química , Pareamento de Bases/genética , Substâncias Intercalantes/química , Ligantes , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleotídeos/química , Nucleotídeos/genética , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
13.
Nucleic Acids Res ; 48(15): 8461-8473, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633759

RESUMO

DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA/genética , Nucleotidiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Estruturas Cromossômicas/efeitos dos fármacos , Estruturas Cromossômicas/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/genética , Complexos Multiproteicos/genética , Compostos de Mostarda Nitrogenada/farmacologia , Saccharomyces cerevisiae/genética
14.
Hum Genet ; 139(12): 1499-1511, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32533362

RESUMO

Vascular anomalies (VAs), comprising wide subtypes of tumors and malformations, are often caused by variants in multiple tyrosine kinase (TK) receptor signaling pathways including TIE2, PIK3CA and GNAQ/11. Yet, a portion of individuals with clinical features of VA do not have variants in these genes, suggesting that there are undiscovered pathogenic factors underlying these patients and possibly with overlapping phenotypes. Here, we identified one rare non-synonymous variant (c.968A > G) in the seventh exon of GPAA1 (Glycosylphosphatidylinositol Anchor Attachment Protein 1), shared by the four affected members of a large pedigree with multiple types of VA using whole-exome sequencing. GPAA1 encodes a glycosylphosphatidylinositol (GPI) transamidase complex protein. This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum (ER). We showed such variant led to scarce expression of GPAA1 protein in vascular endothelium and induced a localization change from ER membrane to cytoplasm and nucleus. In addition, expressing wild-type GPAA1 in endothelial cells had an effect to inhibit cell proliferation and migration, while expressing variant GPAA1 led to overgrowth and overmigration, indicating a loss of the quiescent status. Finally, a gpaa1-deficient zebrafish model displayed several types of developmental defects as well as vascular dysplasia, demonstrating that GPAA1 is involved in angiogenesis and vascular remodeling. Altogether, our results indicate that the rare coding variant in GPAA1 (c.968A > G) is causally related to familial forms of VAs.


Assuntos
Exoftalmia/genética , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/genética , Malformações Vasculares/genética , Aciltransferases/genética , Adulto , Animais , Sistemas CRISPR-Cas/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Éxons/genética , Exoftalmia/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Malformações Vasculares/patologia , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
15.
Proc Natl Acad Sci U S A ; 117(27): 15650-15658, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571937

RESUMO

Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Lipídeos/química , Nucleoproteínas/química , RNA/química , Anisotropia , Proteínas Intrinsicamente Desordenadas/genética , Lipídeos/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleoproteínas/genética , Pinças Ópticas , Transição de Fase , RNA/genética
16.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
17.
Mol Cell ; 79(4): 603-614.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32579943

RESUMO

Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Livre de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , Coelhos , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , Ubiquitinação
18.
Mol Cell ; 79(4): 546-560.e7, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32589964

RESUMO

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.


Assuntos
Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Fatores de Iniciação de Peptídeos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Nat Struct Mol Biol ; 27(7): 668-677, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541898

RESUMO

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.


Assuntos
Complexos Multiproteicos/química , RNA Polimerase II/metabolismo , Fatores de Transcrição/química , Regulação Alostérica , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , RNA Polimerase II/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo
20.
Genes Dev ; 34(11-12): 819-831, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32354834

RESUMO

Condensin mediates chromosome condensation, which is essential for proper chromosome segregation during mitosis. Prior to anaphase of budding yeast, the ribosomal DNA (RDN) condenses to a thin loop that is distinct from the rest of the chromosomes. We provide evidence that the establishment and maintenance of this RDN condensation requires the regulation of condensin by Cdc5p (polo) kinase. We show that Cdc5p is recruited to the site of condensin binding in the RDN by cohesin, a complex related to condensin. Cdc5p and cohesin prevent condensin from misfolding the RDN into an irreversibly decondensed state. From these and other observations, we propose that the spatial regulation of Cdc5p by cohesin modulates condensin activity to ensure proper RDN folding into a thin loop. This mechanism may be evolutionarily conserved, promoting the thinly condensed constrictions that occur at centromeres and RDN of mitotic chromosomes in plants and animals.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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