N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective ‘reader’ proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

Summary N 6 -methyladenosine (m 6 A), the most prevalent internal RNA modification on mammalian messenger RNAs (mRNAs), regulates fates and functions of modified transcripts through m 6 A-specific binding proteins 1 – 5 . m 6 A is abundant in the nervous system and modulates various neural functions 6 – 11 . While m 6 A marks groups of mRNAs for coordinated degradation in various physiological processes 12 – 15 , the relevance of m 6 A in mRNA translation remains largely unknown in vivo . Here we show that, through its binding protein Ythdf1, m 6 A promotes protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 ( Ythdf1 -KO) exhibit learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Ythdf1 re-expression in the hippocampus of adult Ythdf1 -KO mice rescues behavioral and synaptic defects, while hippocampus-specific acute knockdown of Ythdf1 or Mettl3 , the catalytic component of m 6 A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of Ythdf1 binding sites and m 6 A sites on hippocampal mRNAs uncovered key neuronal genes. Nascent protein labeling and tether reporter assays in hippocampal neurons revealed that Ythdf1 enhances protein synthesis in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m 6 A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli.

BackgroundWhat was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY’s effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.MethodsMice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.ResultsMice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.ConclusionThese findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.

Tight junctions (TJs) are fundamental features of both epithelium and endothelium and are indispensable for vertebrate organ formation and homeostasis. However, mice lackingOccludin(Ocln) develop relatively normally to term. Here we show thatOclnis essential for mammary gland physiology, as mutant mice fail to produce milk. Surprisingly,Oclnnull mammary glands showed intact TJ function and normal epithelial morphogenesis, cell differentiation, and tissue polarity, suggesting thatOclnis not required for these processes. Using single-cell transcriptomics, we identified milk-producing cells (MPCs) and found they were progressively more prone to endoplasmic reticulum (ER) stress as protein production increased exponentially during late pregnancy and lactation. Importantly,Oclnloss in MPCs resulted in greatly heightened ER stress; this in turn led to increased apoptosis and acute shutdown of protein expression, ultimately leading to lactation failure in the mutant mice. We show that the increased ER stress was caused by a secretory failure of milk proteins inOclnnull cells. Consistent with an essential role in protein secretion, Occludin was seen to reside on secretory vesicles and to be bound to SNARE proteins. Taken together, our results demonstrate thatOclnprotects MPCs from ER stress by facilitating SNARE-dependent protein secretion and raise the possibility that other TJ components may participate in functions similar toOcln.

Salvianolic acid B (Sal B) is a water-soluble compound found in the traditional Chinese medicine, Radix Salviae miltiorrhizae, and has been widely used to treat a variety of diseases in Asian cultures. Sal B was shown to inhibit apoptosis in many cell types, but its effect on hepatocyte apoptosis is unknown. In this study, we attempt to show that Sal B attenuates hepatocyte apoptosis and hepatic injury induced by lipopolysaccharide and D-galactosamine in mice. Sal B also inhibits apoptosis that is induced by the death receptor in the HL-7702 hepatocyte cell line. Apoptosis in vitro is determined by flow cytometry, DNA electrophoresis and high content screening assay. The antiapoptotic effect is generated by reducing the expression of tumor necrosis factor alpha receptor type 1, balancing the expression of Bcl-2 family members, decreasing the release of cytochrome C from the mitochondria into the cytosol and inhibiting activated Caspase-3. These findings suggest that Sal B can effectively inhibit hepatocyte apoptosis as well as the underlying mechanisms related to regulating mediators in death receptor and mitochondrial pathways.

Autism spectrum disorder (ASD) is a neurodevelopmental disease with a strong heritability, but recent evidence suggests that epigenetic dysregulation may also contribute to the pathogenesis of ASD. Especially, increased methylation at the MECP2 promoter and decreased MECP2 expression were observed in the brains of ASD patients. However, the causative relationship of MECP2 promoter methylation and ASD has not been established. In this study, we achieved locus-specific methylation at the transcription start site (TSS) of Mecp2 in Neuro-2a cells and in mice, using nuclease-deactivated Cas9 (dCas9) fused with DNA methyltransferase catalytic domains, together with five locus-targeting sgRNAs. This locus-specific epigenetic modification led to a reduced Mecp2 expression and a series of behavioral alterations in mice, including reduced social interaction, increased grooming, enhanced anxiety/depression, and poor performance in memory tasks. We further found that specifically increasing the Mecp2 promoter methylation in the hippocampus was sufficient to induce most of the behavioral changes. Our finding therefore demonstrated for the first time the casual relationship between locus-specific DNA methylation and diseases symptoms in vivo, warranting potential therapeutic application of epigenetic editing.

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