Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation

m⁶A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m⁶A-modified base. The recruitment of the mRNA m⁶A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Approximately one-fourth of eukaryotic mRNAs harbor at least one m6A-modified base, but how is this regulated? This study shows that cells can use liquid-liquid phase separation to regulate dynamic assembly of the mRNA m6A methyltransferase complex (METTL3/METTL14/WTAP), with stoichiometries that depend on condensate partitioning in a substrate binding-dependent manner.     

LB克隆系统的构建及在鞘氨醇单胞菌基因融合表达中的应用

为摆脱限制性酶切位点不足的限制,构建可灵活改变多基因融合方向的表达载体,基于ⅡS型和ⅡT型限制性内切酶LguⅠ和BbvC Ⅰ设计开发了 LB克隆系统.该克隆系统是以广宿主质粒pBBR1MCS-3为初始载体,利用 PCR的方法,在其多克隆位点区插入LB片段(GCTCTTCCTCAGC)构建得到的.LB片段含Lgu Ⅰ和B...     

Effects of Radix Polygalae on Cognitive Decline and Depression in Estradiol Depletion Mouse Model of Menopause

Postmenopausal syndrome refers to symptoms caused by the gradual decrease in female hormones after mid-40 years. As a target organ of estrogen, decrease in estrogen causes various changes in brain function such as a decrease in choline acetyltransferase and brain-derived neurotrophic factor; thus, postmenopausal women experience cognitive decline and more depressive symptoms than age-matched men. Radix Polygalae has been used for memory boosting and as a mood stabilizer and its components have shown neuroprotective, antidepressant, and stress relief properties. In a mouse model of estrogen depletion induced by 4-vinylcyclohexene diepoxide, Radix Polygalae was orally administered for 3 weeks. In these animals, cognitive and depression-related behaviors and molecular changes related to these behaviors were measured in the prefrontal cortex and hippocampus. Radix Polygalae improved working memory and contextual memory and despair-related behaviors in 4-vinylcyclohexene diepoxide-treated mice without increasing serum estradiol levels in this model. In relation to these behaviors, choline acetyltransferase and brain-derived neurotrophic factor in the prefrontal cortex and hippocampus and bcl-2-associated athanogene expression increased in the hippocampus. These results implicate the possible benefit of Radix Polygalae in use as a supplement of estrogen to prevent conditions such as postmenopausal depression and cognitive decline.     

A Passive Anti-icing Strategy Based on a Superhydrophobic Mesh with Extremely Low Ice Adhesion Strength

Although superhydrophobic materials have attracted much research interest in anti-icing,some controversy still exists.In this research,we report a cost-effective method used to verify the contribution of area fraction to ice adhesion strength.We tried to partially-embed siliea nanopnarticles into microscale fabrics of a commercial polyamide mesh.Then,the area fraction could be determined by altering the mesh size.Generally,the ice adhesion strength decreases as the area fraction decreases.An ice adhesion strength of~1.9 kPa and a delayed freezing time of~1048 s can be obtained.We attribute the low ice adhesion strength to the combination of superhydro-phobicity and stress concentration.The superhydrophobicity prohibits the water from penetrating into the voids of the meshes,and the small actual contact area leads to stress concentration which promotes interfacial crack propagation.Moreover,our superhydrophobic mesh simultaneously exhibis a micro-nano hierarchical structure and a partally-cmbedded structure.Therefore,the as-prepared superhydrophobic mesh retained the ieephobicity after 20 icingldeicing cycles,and maintained its superhydrophobicity even afier 60 sandpaper-abrasion cycles and a 220"C thermal treatment.     

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Rab31 promotes the invasion and metastasis of cervical cancer cells by inhibiting MAPK6 degradation

Persistent infection with high-risk human papillomavirus (HPV) is the main risk factor for cervical cancer. Our mass spectrometry data showed that the Ras-associated binding protein Rab31 was upregulated by HPV; however, little is known regarding the role of Rab31 in the metastasis of cervical cancer cells. In this study, we showed that Rab31 was highly expressed in cervical cancer tissues and cells, and both HPV E6 and E7 promoted the expression of Rab31. Rab31 knockdown inhibited while Rab31 overexpression promoted the migration and invasion capabilities of cervical cancer cells. Additionally, Rab31 knockdown inhibited the epithelial-mesenchymal transition (EMT) and cytoskeletal rearrangement in cervical cancer cells. Furthermore, Rab31 interacted with mitogen-activated protein kinase 6 (MAPK6), and Rab31 knockdown inhibited the expression of MAPK6, which was mainly localized in the cytoplasm. More importantly, Rab31 knockdown promoted and Rab31 overexpression inhibited MAPK6 degradation. Accordingly, MAPK6 overexpression restored the decreased migration potential caused by Rab31 knockdown. Finally, a xenograft mouse model showed that Rab31 knockdown in cervical cancer cells led to reduced tumor growth and impaired lung and liver metastasis in vivo. In conclusion, Rab31 plays a crucial role in cervical cancer metastasis by inhibiting MAPK6 degradation. Thus, Rab31 may serve as a novel therapeutic target to manage cervical cancer.     

Seasonal expressions of GPR41 and GPR43 in the colon of the wild ground squirrels (Spermophilus dauricus)

G-protein-coupled receptor 41 (GPR41) and G-protein-coupled receptor 43 (GPR43) are important short-chain fatty acids (SCFAs) receptors. Previous studies indicated that GPR41 and GPR43 are involved in the secretion of gastrointestinal peptides, and glucose and lipid metabolism, and are closely related to obesity and type II diabetes, and other diseases. The purpose of the study was to explore the relationship of the GPR41 and GPR43 with seasonal breeding, and provide new prospects for further exploring the nutritional needs of breeding. We identified the localization and expression levels of GPR41 and GPR43 in the colon of the wild ground squirrels (Spermophilus dauricus) both in the breeding season and non-breeding season. The histological results revealed that the lumen diameter of the colon had obvious seasonal changes, and the diameter of the colonic lumen in the non-breeding season was larger than that in the breeding season. Immunohistochemical staining suggested that GPR41 and GPR43 are expressed in the simple layer columnar epithelium. In addition, compared with the breeding season, the mRNA and protein expression levels of GPR41 and GPR43 in the colon were higher during the non-breeding season. In general, these results indicated that GPR41 and GPR43 might play a certain role in regulating seasonal breeding.Key words: GPR41, GPR43, colon, wild ground squirrel, seasonal breeding     

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson’s disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first “mtISR suppressor” localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.Subject terms: Stress signalling, Mitochondria