N6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

N 6 -methyladenosine (m 6 A), the most abundant internal modification on mRNAs in eukaryotes, play roles in adipogenesis. However, the underlying mechanism remains largely unclear. Here, we show that m 6 A plays a critical role in regulating macroautophagy/autophagy and adipogenesis through targeting Atg5 and Atg7. Mechanistically, knockdown of FTO, a well-known m 6 A demethylase, decreased the expression of ATG5 and ATG7, leading to attenuation of autophagosome formation, thereby inhibiting autophagy and adipogenesis. We proved that FTO directly targeted Atg5 and Atg7 transcripts and mediated their expression in an m 6 A-dependent manner. Further study identified that Atg5 and Atg7 were the targets of YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). Upon FTO silencing, Atg5 and Atg7 transcripts with higher m 6 A levels were captured by YTHDF2, which resulted in mRNA degradation and reduction of protein expression, thus alleviating autophagy and adipogenesis. Furthermore, we generated an adipose-selective fto knockout mouse and find that FTO deficiency decreased white fat mass and impairs ATG5-and ATG7-dependent autophagy in vivo. Together, these findings unveil the functional importance of the m 6 A methylation machinery in autophagy and adipogenesis regulation, which expands our understanding of such interplay that is essential for development of therapeutic strategies in the prevention and treatment of obesity.

For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.

No abstract

No abstract

No abstract

No abstract

In the present study, a new form of selenium nanoparticle (biogenic nanoselenium (BNS) particles) was synthesized using bacteria. The protection of BNS particles against oxidative stress-induced intestinal barrier dysfunction and the inherent mechanisms of this process were investigated, and selenomethionine (SeMet) and chemically synthesized nanoselenium (Nano-Se) particles were used for comparison. Characterization of BNS particles revealed that they were monodispersed and homogeneous spheres, with an average size of 139.43 ± 7.44 nm. In the mouse model of intestinal oxidative stress, BNS particles were found to protect the mouse intestinal barrier function and preserve intestinal redox homeostasis more efficiently than SeMet and Nano-Se. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells verified the stronger epithelial barrier-protecting effect of BNS particles against oxidative stress, with reduced cell apoptosis and an improved cell redox state. BNS activated the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and increased the expression of its downstream genes, including thioredoxin reductase (TXNRD)-1, NADPH dehydrogenase (NQO)-1, heme oxygenase (HO)-1, and thioredoxin (Trx), in dose- and time-dependent manners. In contrast, SeMet and Nano-Se merely enhanced the activity of the selenoenzymes TXNRD-1 and glutathione peroxidase (GPx)-1, indicating the role of selenium donors. Moreover, the knock down of Nrf2 significantly blocked the antioxidative effect of BNS, confirming that BNS protects the intestinal barrier from oxidative stress-induced damage by activating Nrf2 and its downstream genes. Our results suggest that BNS is a promising selenium species with potential application in treating oxidative stress-related intestinal diseases.