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1.
Cell ; 185(12): 2132-2147.e26, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35688134

RESUMO

RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.


Assuntos
Exossomos , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA/metabolismo , Estabilidade de RNA
2.
Cell ; 179(1): 282-282.e1, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31539497

RESUMO

The RNA exosome is a 3' to 5' ribonuclease that plays a fundamental role in maturation, quality control, and turnover of nearly all types of RNA produced in eukaryotic cells. Here, we present an overview of the structure, composition, and functions of the RNA exosome, including various cytoplasmic and nuclear exosome co-factors and associated protein complexes. To view this SnapShot, open or download the PDF.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , RNA Helicases/metabolismo , Estabilidade de RNA
3.
Cell ; 173(7): 1663-1677.e21, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29906447

RESUMO

The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.


Assuntos
DNA Helicases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/metabolismo , RNA/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Humanos , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , RNA/genética , RNA Helicases/química , Estabilidade de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade por Substrato
4.
Genes Dev ; 36(3-4): 180-194, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058317

RESUMO

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.


Assuntos
Meiose , RNA Helicases , Animais , Regulação da Expressão Gênica , Masculino , Mamíferos/genética , Meiose/genética , Camundongos , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética
5.
Proc Natl Acad Sci U S A ; 115(24): E5506-E5515, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29844170

RESUMO

The nuclear exosome-targeting (NEXT) complex functions as an RNA exosome cofactor and is involved in surveillance and turnover of aberrant transcripts and noncoding RNAs. NEXT is a ternary complex composed of the RNA-binding protein RBM7, the scaffold zinc-knuckle protein ZCCHC8, and the helicase MTR4. While RNA interactions with RBM7 are known, it remains unclear how NEXT subunits collaborate to recognize and prepare substrates for degradation. Here, we show that MTR4 helicase activity is enhanced when associated with RBM7 and ZCCHC8. While uridine-rich substrates interact with RBM7 and are preferred, optimal activity is observed when substrates include a polyadenylated 3' end. We identify a bipartite interaction of ZCCHC8 with MTR4 and uncover a role for the conserved C-terminal domain of ZCCHC8 in stimulating MTR4 helicase and ATPase activities. A crystal structure reveals that the ZCCHC8 C-terminal domain binds the helicase core in a manner that is distinct from that observed for Saccharomyces cerevisiae exosome cofactors Trf4p and Air2p. Our results are consistent with a model whereby effective targeting of substrates by NEXT entails recognition of elements within the substrate and activation of MTR4 helicase activity.


Assuntos
Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Exossomos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Modelos Moleculares , Ligação Proteica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo
6.
Hum Mol Genet ; 26(20): 4028-4041, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29016861

RESUMO

DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson's disease (PD), but growing evidence suggests involvement of DJ-1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH-SY5Y neuroblastoma cell lines that DJ-1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ-1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ-1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ-1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ-1 complexes. In the KD model, the absence of DJ-1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re-expression of DJ-1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7-related PD. Notably, oxidised DJ-1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.


Assuntos
Catecolaminas/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Dopamina/metabolismo , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo
8.
J Am Chem Soc ; 135(43): 15974-7, 2013 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-24144264

RESUMO

The Parkinsonism-associated protein DJ-1 has been suggested to activate the Cu-Zn superoxide dismutase (SOD1) by providing its copper cofactor. The structural and chemical means by which DJ-1 could support this function is unknown. In this study, we characterize the molecular interaction of DJ-1 with Cu(I). Mass spectrometric analysis indicates binding of one Cu(I) ion per DJ-1 homodimer. The crystal structure of DJ-1 bound to Cu(I) confirms metal coordination through a docking accessible biscysteinate site formed by juxtaposed cysteine residues at the homodimer interface. Spectroscopy in crystallo validates the identity and oxidation state of the bound metal. The measured subfemtomolar dissociation constant (Kd = 6.41 × 10(-16) M) of DJ-1 for Cu(I) supports the physiological retention of the metal ion. Our results highlight the requirement of a stable homodimer for copper binding by DJ-1. Parkinsonism-linked mutations that weaken homodimer interactions will compromise this capability.


Assuntos
Cobre/química , Cisteína/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação/fisiologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Sítios de Ligação , Cobre/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Metais/química , Modelos Moleculares , Proteínas Oncogênicas/metabolismo , Conformação Proteica , Proteína Desglicase DJ-1 , Espectrometria de Massas por Ionização por Electrospray , Superóxido Dismutase/química , Superóxido Dismutase-1
9.
Methods Enzymol ; 673: 453-473, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35965016

RESUMO

The nuclear RNA exosome collaborates with the MTR4 helicase and RNA adaptor complexes to process, surveil, and degrade RNA. Here we outline methods to characterize RNA translocation and strand displacement by exosome-associated helicases and adaptor complexes using fluorescence-based strand displacement assays. The design and preparation of substrates suitable for analysis of helicase and decay activities of reconstituted MTR4-exosome complexes are described. To aid structural and biophysical studies, we present strategies for engineering substrates that can stall helicases during translocation, providing a means to capture snapshots of interactions and molecular steps involved in substrate translocation and delivery to the exosome.


Assuntos
Exossomos , Proteínas de Saccharomyces cerevisiae , DNA Helicases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Humanos , Oligonucleotídeos/metabolismo , RNA/metabolismo , RNA Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Elife ; 72018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29360036

RESUMO

Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, 'ketu', caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3'→5' RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Germinativas/fisiologia , Meiose , RNA Helicases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Testes Genéticos , Infertilidade , Masculino , Camundongos , Mutação de Sentido Incorreto , RNA Helicases/genética
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