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.

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.

SignificanceAberrant or unwanted transcripts can be degraded by the RNA exosome with the help of the nuclear exosome-targeting (NEXT) complex. NEXT, composed of RNA-binding protein RBM7, scaffold ZCCHC8, and helicase MTR4, is implicated in stress response, neurodegeneration, and viral ribogenesis. Here, we characterize the activities of NEXT that support its role in exosome-mediated decay. NEXT catalyzes 3′→5′ helicase activity and disrupts RNA:RNA and DNA:RNA duplexes more efficiently than MTR4. Optimal activity is observed when substrates include a uridine-rich motif, for interactions with RBM7, and a 3′ poly(A) tail. The ZCCHC8 C-terminal domain binds the helicase core and can stimulate MTR4 helicase/ATPase activities. Our results highlight the interplay among NEXT subunits to ensure effective targeting of substrates.

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 3ʹ→5ʹ RNA helicase activity in vitro, and has similarity within its YTH domain to an N 6 -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. Version 2: The following experimental changes were incorporated:• RNA-seq analyses of wild-type and Ythdc2 mutant testes at 8, 9, and 10 dpp (i.e., during the germline transition into meiosis).• Purification and characterization of recombinant YTHDC2 protein, demonstrating RNA helicase activity and effect of the ketu mutation.• Expanded histological and cytological analyses (DMC1-staining; PAS-staining and TUNEL assay at 8 dpp; further pH3 and tubulin staining along with SYCP3 staining in abnormal metaphases to evaluate premature metaphase entry; BrdU incorporation to evaluate premeiotic DNA replication; and more complete evaluation of persistent CCNA2 expression), including quantification of stainings at multiple ages (pH3, α-tubulin, BrdU, SYCP3, and CCNA2 stainings as well as TUNEL assay). Cem Meydan, Nathalie Lailler, Christopher E. Mason, and Christopher D. Lima were added as coauthors.

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.

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin ␤2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis. Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by a relatively selective loss of dopaminergic (DA) neurons and the presence of Lewy bodies in the substantia nigra. The majority of PD cases are sporadic; however, early-onset PD, often monogenic in nature, accounts for between 5 and 10% of all cases (1). Autosomal recessive forms of early-onset PD are caused by mutations in parkin (PARK2) (2), PTEN-induced putative kinase 1 (PARK6; PINK1) (3), and Parkinson protein 7 (PARK7; DJ-1) (4).DJ-1 belongs to the Thi/PfpI protein superfamily and is expressed in a variety of tissues, including the brain (5). DJ-1 has an atypical peroxiredoxin-like peroxidase activity that is involved in scavenging mitochondrial H 2 O 2 , and DJ-1 knockout mice have increased mitochondrial H 2 O 2 levels (6, 7). DJ-1 shifts toward a more acidic isoelectric point in response to reactive oxygen species (ROS) (8, 9), and DJ-1 knockout models show increased sensitivity toward oxidative stress/mitochondrial toxins (9-13). Conversely, overexpression of wild-type (wt) DJ-1 protects cells against ROS/mitochondrial toxins (9, 14). DJ-1 harbors three cysteine residues (C46, C53, and C106); C106 oxidizes to form a cysteine-sulfinic acid, and C106 mutations lead to DJ-1 loss of function (7,10,15).DJ-1 localizes to the cytoplasm, nucleus, and mitochondria (7,16,17). Mitochondrial DJ-1 translocation is enhanced by oxidative stress, and C106 oxidation is necessary for translocation (7). Targeting of DJ-1 to mitochondria protects cells against oxidative stress (18), a...

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.

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.