N6-methyladenine DNA modification in Drosophila.

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.

Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination.

AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. Here, we unexpectedly discover that fly and human AGO proteins, which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P2. The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, our study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response.

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.

N6-methyladenine functions as a potential epigenetic mark in eukaryotes.

N(6)-methyladenine (6mA) is one of the most abundant types of DNA methylation, and plays an important role in bacteria; however, its roles in higher eukaryotes, such as plants, insects, and mammals, have been considered less important. Recent studies highlight that 6mA does indeed occur, and that it plays an important role in eukaryotes, such as worm, fly, and green algae, and thus the regulation of 6mA has emerged as a novel epigenetic mechanism in higher eukaryotes. Despite this intriguing development, a number of important issues regarding its biological roles are yet to be addressed. In this review, we focus on the 5mC and 6mA modifications in terms of their production, distribution, and the erasure of 6mA in higher eukaryotes including mammals. We perform an analysis of the potential functions of 6mA, hence widening understanding of this new epigenetic mark in higher eukaryotes, and suggesting future studies in this field.

Activation of Smurf E3 ligase promoted by smoothened regulates hedgehog signaling through targeting patched turnover.

Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.

The TET-Sall4-BMP regulatory axis controls craniofacial cartilage development.

Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here, we employ the zebrafish model to investigate mechanisms of CFM pathogenesis. In early embryos, tet2 and tet3 are essential for pharyngeal cartilage development. Single-cell RNA sequencing reveals that loss of Tet2/3 impairs chondrocyte differentiation due to insufficient BMP signaling. Moreover, biochemical and genetic evidence reveals that the sequence-specific 5mC/5hmC-binding protein, Sall4, binds the promoter of bmp4 to activate bmp4 expression and control pharyngeal cartilage development. Mechanistically, Sall4 directs co-phase separation of Tet2/3 with Sall4 to form condensates that mediate 5mC oxidation on the bmp4 promoter, thereby promoting bmp4 expression and enabling sufficient BMP signaling. These findings suggest the TET-BMP-Sall4 regulatory axis is critical for pharyngeal cartilage development. Collectively, our study provides insights into understanding craniofacial development and CFM pathogenesis.

Argonaute-dependent ribosome-associated protein quality control.

Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a key regulator of RQC that targets substrates for ubiquitination. Argonaute proteins (AGOs) are central players in miRNA-mediated gene silencing and have recently been shown to also regulate RQC by facilitating Ltn1. Therefore, AGOs directly coordinate post-transcriptional gene silencing and RQC, ensuring efficient gene silencing. We summarize the principles of RQC and the functions of AGOs in miRNA-mediated gene silencing, and discuss how AGOs associate with the endoplasmic reticulum (ER) to assist Ltn1 in controlling RQC. We highlight that RQC not only eliminates defective nascent polypeptides but also removes unwanted protein products when AGOs participate.

PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling.

TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells, and genetic experiments show that Ptk2b-deficient mice are more susceptible to viral infection than control mice. Mechanistically, we demonstrate that PTK2B directly phosphorylates residue Tyr591 of TBK1, which increases TBK1 oligomerization and activation. In addition, we find that PTK2B also interacts with the stimulator of interferon genes (STING) and can promote its oligomerization in a kinase-independent manner. Collectively, PTK2B enhances the oligomerization of TBK1 and STING via different mechanisms, subsequently regulating STING-TBK1 activation to ensure efficient antiviral innate immune responses.

Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells.

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).

A protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles.

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).

Single-cell transcriptomic analysis of honeybee brains identifies vitellogenin as caste differentiation-related factor.

The honeybee (Apis mellifera) is a well-known eusocial insect. In honeybee colonies, thousands of sterile workers, including nurse and forager bees, perform various tasks within or outside the hive, respectively. The queen is the only fertile female and is responsible for reproduction. The queen and workers share similar genomes but occupy different caste statuses. We established single-cell transcriptomic atlases of brains from queens and worker subcastes and identified five major cell groups: Kenyon, optic lobe, olfactory projection, glial, and hemocyte cells. By dividing Kenyon and glial cells into multiple subtypes based on credible markers, we observed that vitellogenin (vg) was highly expressed in specific glial-cell subtypes in brains of queens. Knockdown of vg at the early larval stage significantly suppressed the development into adult queens. We demonstrate vg expression as a "molecular signature" for the queen caste and suggest involvement of vg in regulating caste differentiation.

Dynamic FMR1 granule phase switch instructed by m6A modification contributes to maternal RNA decay.

Maternal RNA degradation is critical for embryogenesis and is tightly controlled by maternal RNA-binding proteins. Fragile X mental-retardation protein (FMR1) binds target mRNAs to form ribonucleoprotein (RNP) complexes/granules that control various biological processes, including early embryogenesis. However, how FMR1 recognizes target mRNAs and how FMR1-RNP granule assembly/disassembly regulates FMR1-associated mRNAs remain elusive. Here we show that Drosophila FMR1 preferentially binds mRNAs containing m6A-marked "AGACU" motif with high affinity to contributes to maternal RNA degradation. The high-affinity binding largely depends on a hydrophobic network within FMR1 KH2 domain. Importantly, this binding greatly induces FMR1 granule condensation to efficiently recruit unmodified mRNAs. The degradation of maternal mRNAs then causes granule de-condensation, allowing normal embryogenesis. Our findings reveal that sequence-specific mRNAs instruct FMR1-RNP granules to undergo a dynamic phase-switch, thus contributes to maternal mRNA decay. This mechanism may represent a general principle that regulated RNP-granules control RNA processing and normal development.

PCBP2 maintains antiviral signaling homeostasis by regulating cGAS enzymatic activity via antagonizing its condensation.

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.

Understanding autism spectrum disorders with animal models: applications, insights, and perspectives.

Autism spectrum disorder (ASD) is typically characterized by common deficits in social skills and repetitive/stereotyped behaviors. It is widely accepted that genetic and environmental factors solely or in combination cause ASD. However, the underlying pathogenic mechanism is unclear due to its highly heterogeneous nature. To better understand the pathogenesis of ASD, various animal models have been generated, which can be generally divided into genetic, environment-induced, and idiopathic animal models. In this review, we summarize the common animals used for ASD study and then discuss the applications, clinical insights, as well as challenges and prospects of current ASD animal models.

[Wavelength conversion and spectral analysis in periodically polarized lithium niobate waveguide].

Wavelength conversion exploiting cascaded second harmonic and difference frequency generation (c(SHG/DFG)) in periodically polarized LiNbO3 (PPLN) waveguides was experimentally researched. While wavelength converter was pumped with a pulsed wave, the pump pulse can be used to carry the information and wavelength conversion occurs between the pump wave and converted wave, thus wavelength conversion transferring the information from the pump wave to the converted waves includes two processes of second order nonlinear reaction: the first wavelength conversion from pump wave to SH wave occurs with SHG process, and the second wavelength conversion from SH wave to converted wave occurs with DFG process. In the first process the group velocities mismatching (GVM) for pulses at different wavelengths due to material property load the temporal walk-off between pump pulse and SH pulse located in the 1.5 microm band and in the 0.8 microm band, respectively, so that SH pulse slowly propagates compared with pump pulse, and SH pulse width is broadened along propagation length. As a result, in the second process the converted DF pulse generates waveform distortion owing to the broadening of SH pulse in the first process. Both the waveform and the spectrum of converted pulse in our experimental results testify to the fact that SH pulse possesses a narrow spectral width, which is consistent with a long SH pulse, and the spectral width of converted DF pulse is compressed but its temporal width is broadened correspondingly. Therefore the influence of walk-off between pulses demonstrates that the pulsed pumping wavelength conversion is disadvantageous to the transparence of the data format. However, pulsed pumping wavelength conversion also presents great potential that can be applied in future optical networks. Tunable wavelength conversion can be easily implemented by changing the wavelength of control CW, and single-to-multiple channel wavelength conversion can be realized by increasing the number of the CW lasing pump channels. This is very important and it enhances the flexibility in the management of the multi-channel WDM network. Finally, a tunable and single-to-dual channel wavelength converter based on the scheme of pulsed pumping wavelength conversion achieved by our experiment setup, and two channel converted pulses simultaneously replicate the bit rate carried on pump pulses. It is pointed out that the quality such the signal-to-noise ratio of converted pulse is affected by spectral width of control CW.

Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.

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.