Porcine reproductive and respiratory syndrome virus (PRRSV) is known to cause reproductive disorders, such as abortion, in pregnant sows as well as immunosuppressive respiratory complications, leading to severe respiratory tract infections in young pigs. In this study, an in-depth analysis of the miRNomes in mock- and virus-infected pig lungs was carried out. We found that highly expressed ssc-miR-30d-R_1 was decreased in infected lungs, and reduced levels were significantly correlated with infection by PRRSV. Moreover, ssc-miR-30d-R_1 was shown to target Toll-like receptor 4 (TLR4) and to suppress the production of immune cytokines through inhibition of the TLR4/MyD88/NF-κB pathway. ssc-miR-30d-R_1 significantly reduced viral infections and pathological changes in pig lungs in vivo. Our current study reveals the miRNomes of PRRSV-infected pig lungs and indicates that ssc-miR-30d-R_1 is potential therapeutic agent for controlling PRRSV infection.

Stimulator of interferon genes (STING, also known as MITA and ERIS) is critical in protecting the host against DNA pathogen invasion. However, the molecular mechanism underlying the regulation of STING remains unclear. Here, we show that PPM1A negatively regulates antiviral signaling by targeting STING in its phosphatase activity-dependent manner, and in a line with this, PPM1A catalytically dephosphorylates STING and TBK1 in vitro. Importantly, we provide evidence that whereas TBK1 promotes STING aggregation in a phosphorylation-dependent manner, PPM1A antagonizes STING aggregation by dephosphorylating both STING and TBK1, emphasizing that phosphorylation is crucial for the efficient activation of STING. Our findings demonstrate a novel regulatory circuit in which STING and TBK1 reciprocally regulate each other to enable efficient antiviral signaling activation, and PPM1A dephosphorylates STING and TBK1, thereby balancing this antiviral signal transduction.

Innate immunity mediated by Toll signalling has been extensively studied, but how Toll signalling is precisely controlled in balancing innate immune responses remains poorly understood. It was reported that the plasma membrane localization of Drosophila MyD88 is necessary for the recruitment of cytosolic adaptor Tube to the cell surface, thus contributing to Toll signalling transduction. Here we demonstrate that Drosophila Pellino functions as a negative regulator in Toll-mediated signalling. We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner. Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation. Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.

Caveolin1 (CAV1) is involved in lipid homeostasis and endocytosis, but little is known about the significance of CAV1 in the pathogenesis and development of nonalcoholic fatty liver disease (NAFLD). This study aimed to determine the role of CAV1 in NAFLD.

Accumulation of free fatty acids (FFAs) in hepatocytes induces lipotoxicity, leading to non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate the underlying mechanisms by which FFA contributes to the pathogenesis of NAFLD via the regulation of 3-mercaptopyruvate sulfurtransferase (MPST), a key enzyme that regulates endogenous hydrogen sulfide (H2S) biosynthesis.

Cyclic GMP-AMP synthase (cGAS) plays a major role in detecting pathogenic DNA. It produces cyclic dinucleotide cGAMP, which subsequently binds to the adaptor protein STING and further triggers antiviral innate immune responses. However, the molecular mechanisms regulating cGAS enzyme activity remain largely unknown. Here, we characterize the cGAS-interacting protein Poly(rC)-binding protein 2 (PCBP2), which plays an important role in controlling cGAS enzyme activity, thereby mediating appropriate cGAS-STING signaling transduction. We find that PCBP2 overexpression reduces cGAS-STING antiviral signaling, whereas loss of PCBP2 significantly increases cGAS activity. Mechanistically, we show that PCBP2 negatively regulates anti-DNA viral signaling by specifically interacting with cGAS but not other components. Moreover, PCBP2 decreases cGAS enzyme activity by antagonizing cGAS condensation, thus ensuring the appropriate production of cGAMP and balancing cGAS-STING signal transduction. Collectively, our findings provide insight into how the cGAS-mediated antiviral signaling is regulated.

Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5'- and 3'-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. We recently revealed that the Fused (Fu)/Smurf complex degrades the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. Here we report that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, we show that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Our mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate.

Innate antiviral immunity is the first line of the host defense system that rapidly detects invading viruses. Mitochondria function as platforms for innate antiviral signal transduction in mammals through the adaptor protein, MAVS. Excessive activation of MAVS-mediated antiviral signaling leads to dysfunction of mitochondria and cell apoptosis that likely causes the pathogenesis of autoimmunity. However, the mechanism of how MAVS is regulated at mitochondria remains unknown. Here we show that the Cytochrome c Oxidase (CcO) complex subunit COX5B physically interacts with MAVS and negatively regulates the MAVS-mediated antiviral pathway. Mechanistically, we find that while activation of MAVS leads to increased ROS production and COX5B expression, COX5B down-regulated MAVS signaling by repressing ROS production. Importantly, our study reveals that COX5B coordinates with the autophagy pathway to control MAVS aggregation, thereby balancing the antiviral signaling activity. Thus, our study provides novel insights into the link between mitochondrial electron transport system and the autophagy pathway in regulating innate antiviral immunity.

Genetic analyses have linked microRNA-137 (MIR137) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder. miR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss-of-function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline knockout or nervous system knockout (cKO) leads to postnatal lethality, while heterozygous germline knockout and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, phosphodiesterase 10a (Pde10a), is elevated in heterozygous knockout mice. Treatment with the Pde10a inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1-0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that plays a critical role in regulating antiviral signaling. cGAS binds to DNA and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which is essential for downstream signal transduction. The antiviral response is a rapid biological process; however, cGAS itself has relatively low DNA binding affinity, implying that formation of the cGAS-DNA complex requires an additional factor(s) that promotes cGAS-DNA binding, allowing efficient antiviral signal transduction. Here, we report that the Ku proteins (Ku80 and Ku70) directly interact with cGAS and positively regulate cGAS-mediated antiviral signaling. Mechanistically, we find that the interaction of the Ku proteins with cGAS significantly increases the DNA-binding affinity of cGAS and promotes cGAS condensation in the cytosol, thereby enhancing cGAS catalytic activity. Our results show that the Ku proteins are critical partners of cGAS in sensing DNA virus infection and ensuring efficient innate immune signal transduction.

Here, we present a general protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles (GUVs). In this protocol, we use GUVs to mimic Ago1 protein's phase separation behavior on the membrane through binding with phosphoinositides (PIPs). We provide procedures to prepare fluorescent-labeled Ago1 protein and PI(4,5)P2-containing GUVs, followed by steps to assess Ago1 protein's phase separation in 3D time-lapse images. This protocol can be applied to investigate a membrane-associated protein's behavior on the membrane. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Mitochondrial antiviral signalling protein (MAVS) acts as a critical adaptor protein to transduce antiviral signalling by physically interacting with activated RIG-I and MDA5 receptors. MAVS executes its functions at the outer membrane of mitochondria to regulate downstream antiviral signalling, indicating that the mitochondria provides a functional platform for innate antiviral signalling transduction. However, little is known about whether and how MAVS-mediated antiviral signalling contributes to mitochondrial homeostasis. Here we show that the activation of MAVS is sufficient to induce autophagic signalling, which may mediate the turnover of the damaged mitochondria. Importantly, we find MAVS directly interacts with LC3 through its LC3-binding motif 'YxxI', suggesting that MAVS might act as an autophagy receptor to mediate mitochondrial turnover upon excessive activation of RLR signalling. Furthermore, we provide evidence that both MAVS self-aggregation and its interaction with TRAF2/6 proteins are important for MAVS-mediated mitochondrial turnover. Collectively, our findings suggest that MAVS acts as a potential receptor for mitochondria-associated autophagic signalling to maintain mitochondrial homeostasis.

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.

Somatic cell reprogramming toward induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine. However, the reprogramming process mediated by the traditional defined factors (OSMK) is slow and extremely inefficient. Here, we develop a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system. We show that the efficiency of OySyNyK-induced iPSCs is up to 100-fold higher than the OSNK and the reprogramming by OySyNyK is very rapid and is initiated in 24 hr. We find that OySyNyK factors significantly increase Tet1 expression at the early stage and interact with Tet1/2 to promote reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system but also uncover a mechanism by which engineered factors coordinate with TETs to regulate 5hmC-mediated epigenetic control.

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.

The nuclear transcription factor NF-kappaB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus (KSHV, also known as Human Herpes virus 8)-encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR.

A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. We have previously shown that N6-methyladenine (6mA) DNA modification is highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. Here we report that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. We find that zelda encoding the pioneer factor Zelda is marked by 6mA. Our genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, our findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.