The YTH domain-containing protein Mmi1, together with other factors, constitutes the machinery used to selectively remove meiosis-specific mRNA during the vegetative growth of fission yeast. Mmi1 directs meiotic mRNAs to the nuclear exosome for degradation by recognizing their DSR (determinant of selective removal) motif. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif, demonstrating that the Mmi1 YTH domain selectively recognizes the DSR motif. Intriguingly, Mmi1 also contains a potential m6A (N6-methyladenine)-binding pocket, but its binding of the DSR motif is dependent on a long groove opposite the m6A pocket. The DSR-binding mode is distinct from the m6A RNA-binding mode utilized by other YTH domains. Furthermore, the m6A pocket cannot bind m6A RNA. Our structural and biochemical experiments uncover the mechanism of the YTH domain in binding the DSR motif and help to elucidate the function of Mmi1.

Gene regulatory mechanisms rely on a complex network of RNA processing factors to prevent untimely gene expression. In fission yeast, the highly conserved ortholog of human ERH, called Erh1, interacts with the YTH family RNA binding protein Mmi1 to form the Erh1-Mmi1 complex (EMC) implicated in gametogenic gene silencing. However, the structural basis of EMC assembly and its functions are poorly understood. Here, we present the co-crystal structure of the EMC that consists of Erh1 homodimers interacting with Mmi1 in a 2:2 stoichiometry via a conserved molecular interface. Structure-guided mutation of the Mmi1Trp112 residue, which is required for Erh1 binding, causes defects in facultative heterochromatin assembly and gene silencing while leaving Mmi1-mediated transcription termination intact. Indeed, EMC targets masked in mmi1∆ due to termination defects are revealed in mmi1W112A. Our study delineates EMC requirements in gene silencing and identifies an ERH interface required for interaction with an RNA binding protein.

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.ells respond to DNA double-strand breaks (DSBs) by mounting a series of signal transduction events that culminate in cell arrest and DNA repair (1). Signal transduction entails the coordinated assembly of a cohort of DNA damage mediator proteins, including p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80), at the damaged chromatin, which, in turn, enforces checkpoint control and cell tolerance to DNA damage (2, 3). Paradoxically, although DSB loading of 53BP1 and RAP80 underlies robust activation of DNA damage responses (DDRs), they operate at the expense of high-fidelity DNA repair, because their productive accumulation at DSB-flanking chromatin blocks DNA end resection and suppresses RAD51-dependent homologous recombination (HR) repair (4-11). Exactly how cells achieve a balance between robust DSB signal transduction and HR repair remains to be defined, but several lines of evidence converge on the idea that limiting the extent of DSB ubiquitylation may selectively promote HR repair (12-15), highlighting the need for cell-intrinsic mechanisms to restrain chromatin responses arising from DSBs (16).Intriguingly, one such cell-intrinsic strategy involves ring finger protein 169 (RNF169), a paralog of the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties) syndrome protein RNF168 (17,18). In stark contrast to RNF168, which plays positive roles in amplifying ubiquitin-dependent DSB signals (18,19), RNF169 is endowed with antagonistic properties in the DSB signal transduction pathway. Indeed, RNF169 competes for RNF168-catalyzed ubiquitin adducts and displaces 53BP1 and RAP80 from DSBs (14,20,21). Despite its putative role as an integral component of the DSB signal transduction cascade, how RNF169 is regulated mechanistically and how it contributes to the multipartite self-restraining mechanisms in DNA damage surveillance and...

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo–electron microscopy structures of human HHAT in complex with its palmitoyl–coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

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BackgroundAspirin has been demonstrated to promote osteoblast-mediated bone formation and inhibit osteoclast (OC)-mediated bone resorption. However, it remains unclear whether aspirin influences other immune cells during bone resorption. Dendritic cells (DCs), the most potent antigen-presenting cells, can also transdifferentiate into active OCs in the presence of receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The effects of aspirin on DC-derived OCs (DDOCs) were investigated in the current study.MethodsFlow cytometry and mixed lymphocyte reaction (MLR) assays were used for DC identification. The proliferative capacity of DCs was determined by BrdU assays. Apoptosis was examined by flow cytometry. The osteoclastic potential of DCs was tested using tartrate-resistant acid phosphatase (TRAP) staining, western blotting, and reverse transcription polymerase chain reaction (RT-PCR). Western blotting was also used to examine signaling pathways. A mandibular bone defect model was established to assess the effect of aspirin on bone resorption.ResultsAspirin had no influence on the surface phenotype, proliferation, or apoptosis of DCs, though aspirin significantly inhibited osteoclast differentiation in RANKL-stimulated DCs. DC osteoclast differentiation was modulated by aspirin via the nuclear factor kappa B (NF-κB)/nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) signaling pathway. Aspirin treatment also had favorable therapeutic effects on bone regeneration in the bone defect model, and the number of osteoclasts was decreased.ConclusionsAspirin inhibited RANKL-induced OC differentiation in DCs via the NF-κB pathway, downregulating expression of NFATc1. Aspirin treatment promoted bone regeneration by inhibiting DDOC activation in the early stages of inflammation in a rat mandibular bone defect model.

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Catalytic transfer hydrogenation (CTH) reaction is considered as a potential route for upgrading bio-based carbonyl compounds to their corresponding alcohols.