Human TRMT1 catalyzes m2G or m22G formation on tRNAs in a substrate-dependent manner.

TRMT1 is an N2-methylguanosine (m2G) and N2,N2-methylguanosine (m22G) methyltransferase that targets G26 of both cytoplasmic and mitochondrial tRNAs. In higher eukaryotes, most cytoplasmic tRNAs with G26 carry m22G26, although the majority of mitochondrial G26-containing tRNAs carry m2G26 or G26, suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these tRNAs. Loss-of-function mutations of human TRMT1 result in neurological disorders and completely abrogate tRNA:m22G26 formation. However, the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive, hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1 mutations. Here, we showed that human TRMT1 independently catalyzes formation of the tRNA:m2G26 or m22G26 modification in a substrate-dependent manner, which explains the distinct distribution of m2G26 and m22G26 on cytoplasmic and mitochondrial tRNAs. For human TRMT1-mediated tRNA:m22G26 formation, the semi-conserved C11:G24 serves as the determinant, and the U10:A25 or G10:C25 base pair is also required, while the size of the variable loop has no effect. We defined the requirements of this recognition mechanism as the "m22G26 criteria". We found that the m22G26 modification occurred in almost all the higher eukaryotic tRNAs conforming to these criteria, suggesting the "m22G26 criteria" are applicable to other higher eukaryotic tRNAs.

Effects of Broussonetia papyrifera (L.) L'Hér. ex Vent. fruits water extract on hippocampal neurogenesis in the treatment of APP/PS1 transgenic mice.

Background: Adult neurogenesis plays an important role in repairing damaged neurons and improving cognitive impairment in Alzheimer's disease (AD). B. Papyrifera (L.) L'Hér. ex Vent. fruits (BL), a traditional Chinese medicine for tonifying the kidney, has been reported to improve cognitive function in AD mice, but the underlying mechanisms have not been clearly illuminated. This study aimed to provide an overview of the differential compounds in the brain of APP/PS1 mice after BL water extract (BLWE) treatment through metabolomics technology and to elucidate whether the therapeutic effect and mechanism are through the enhancement of neurogenesis. Methods: APP/PS1 transgenic mice were treated with different doses of BLWE. After 6 weeks of intragastric injection, the therapeutic effects of BLWE on APP/PS1 transgenic mice were determined by the Morris water maze test, immunohistochemistry, hematoxylin & eosin and Nissl staining, enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Subsequently, metabolomics technology was used to analyze the regulatory effect of BLWE on differential compounds in the brain of APP/PS1 mice, and on this basis, its molecular mechanism of BLWE was screened. Finally, the protein expression of the Wnt/ß-catenin signaling pathway was detected by Western blotting. Results: After BLWE treatment, the learning and memory function of APP/PS1 mice were significantly improved, which was related to the increase in the number of Nestin+/BrdU+ and NeuN+/BrdU+ cells, and the decrease in the number of apoptotic cells in the hippocampus. BLWE treatment could also up-regulate the expression of synapse-associated proteins. Moreover, BLWE could modulate endogenous metabolic compounds in the brains of AD mice, including N-acetyl-aspartate, glutamine, etc. Furthermore, BLWE inhibited the phosphorylation of Tyr216-GSK-3ß and ß-catenin protein while increased CyclinD1 protein expression. Conclusion: We demonstrated that BLWE can enhance neural stem cells proliferation and improve neurogenesis, thereby efficiently repairing damaged neurons in the hippocampus and ameliorating cognitive impairment in APP/PS1 transgenic mice. The mechanism is at least partly through activating the Wnt/ß-catenin signaling pathway.

A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription.

Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA-binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2'-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial-mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA-modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.

Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2.

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.

THUMPD3-TRMT112 is a m2G methyltransferase working on a broad range of tRNA substrates.

Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m2G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m2G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3-TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3'-CCA of mature tRNAs. We also showed that m2G7 of tRNATrp was introduced by THUMPD3-TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3-knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m2G6/7 modification and pave a way for further studies of the role of m2G in sperm tRNA derived fragments.

ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.

Kaempferol, a Major Flavonoid in Ginkgo Folium, Potentiates Angiogenic Functions in Cultured Endothelial Cells by Binding to Vascular Endothelial Growth Factor.

Kaempferol is a major flavonoid in Ginkgo Folium and other edible plants, which is being proposed here to have roles in angiogenesis. Angiogenesis is important in both physiological and pathological development. Here, kaempferol was shown to bind with vascular endothelial growth factor (VEGF), probably in the heparin binding domain of VEGF: this binding potentiated the angiogenic functions of VEGF in various culture models. Kaempferol potentiated the VEGF-induced cell motility in human umbilical vein endothelial cells (HUVECs), as well as the sub-intestinal vessel sprouting in zebrafish embryos and formation of microvascular in rat aortic ring. In cultured HUVECs, application of kaempferol strongly potentiated the VEGF-induced phosphorylations of VEGFR2, endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase (Erk) in time-dependent and concentration-dependent manners, and in parallel the VEGF-mediated expressions of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were significantly enhanced. In addition, the potentiation effect of kaempferol was revealed in VEGF-induced migration of skin cell and monocyte. Taken together, our results suggested the pharmacological roles of kaempferol in potentiating VEGF-mediated functions should be considered.

Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Polydatin, also called piceid, is a stilbenoid glucoside of a resveratrol derivative. It derives mainly from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. Although the role of P. cuspidatum root in angiogenesis has been reported, the active chemical or chemicals responsible for such function is not known. Here, polydatin was proposed to bind VEGF, which therefore altered the functions of VEGF in angiogenesis. Several lines of evidence supported the pharmaceutical effects of polydatin in VEGF-induced angiogenesis. In human umbilical vein endothelial cells, polydatin inhibited VEGF-stimulated cell proliferation, cell migration, and tube formation. Moreover, polydatin showed suppressive effects on the subintestinal vessel formation in zebrafish embryos. In signaling cascades, polydatin application attenuated VEGF-induced phosphorylations of VEGF receptor 2 and JNK. Moreover, the VEGF-induced phosphorylations of Akt, eNOS, and Erk were significantly decreased in the presence of polydatin. In parallel, the formation of reactive oxygen species, triggered by VEGF, was markedly decreased under polydatin application. Thus, our results supported the angiogenic roles of polydatin, as well as its signaling mechanism in blocking VEGF-mediated responses. The current study provides support for the possible development of polydatin as a potential therapeutic agent for treatment and prevention of angiogenesis-related diseases.-Hu, W.-H., Wang, H.-Y., Kong, X.-P., Xiong, Q.-P., Poon, K. K.-M., Xu, L., Duan, R., Chan, G. K.-L., Dong, T. T.-X., Tsim, K. W.-K. Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Binding of Resveratrol to Vascular Endothelial Growth Factor Suppresses Angiogenesis by Inhibiting the Receptor Signaling.

Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and red wine, and was identified for its binding to vascular endothelial growth factor (VEGF) by using HerboChips screening. The binding, therefore, resulted in alterations of VEGF binding to its receptor and revealed the roles of VEGF in angiogenesis. Several lines of evidence gave support to the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein endothelial cells (HUVECs), compared with a VEGF-induced group, resveratrol, at a high concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion, and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51%, and 120 ± 10.26%, respectively. Moreover, resveratrol inhibited the subintestinal vessel formation in zebrafish embryo. In signaling cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor 2 phosphorylation and c-Jun N-terminal kinase phosphorylation. Moreover, the VEGF-mediated phosphorylations of endothelial nitric oxide synthase, protein kinase B, and extracellular signal-regulated kinase were obviously decreased by (3 ± 0.37)-, (2 ± 0.27)- and (6 ± 0.23)-fold, respectively, in the presence of resveratrol at high concentration. Parallelly, the VEGF-induced reactive oxygen species formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment. Thus, our results provided support to the antiangiogenic roles of resveratrol, as well as its related signaling mechanisms, in attenuating the VEGF-mediated responses. The present results supported possible development of resveratrol, which should be considered as a therapeutic agent in terms of prevention and clinical treatment of diseases related to angiogenesis.

[Study on preparation and characterization of resveratrol solid lipid nanoparticles and its anticancer effects in vitro].

OBJECTIVE: To prepare resveratrol solid lipid nanoparticles (Res-SLN) and investigate its physical and chemical speciality and anticancer effects in vitro. METHODS: Res-SLN was prepared by the solvent emulsification-evaporation method. Its morphology, particle size and zata potential were examined by transmission electron microscope and laser granularity equipment. Its entrapment efficiency, drug loading, release concentration were determined by HPLC. Its anticancer effect of Res-SLN in vitro were studied by MTT. RESULTS: Res-SLN assumed spherical shape. Its distribution of diameter was even with average particle size of 96. 7 nm, zata potential was--16.3mV, drug loading was (7.95 +/- 0.21)%, entrapment efficiency was (91.34 +/- 0.18)%; Res-SLN could retard drug release in vitro and its cytotoxicity was significantly higher than that of oridonin solution against HepG2 cells. CONCLUSION: Res-SLN has high entrapment efficiency and drug loading, uniform particle size, and can retard drug release in vitro and enhance anticancer effect.

[Preparation of alpha-hydrocycholic-beta-cyclodextrin inclusion compound].

OBJECTIVE: To study the preparation of alpha-Hydrocycholic-beta-Cyclodextrin inclusion compound. METHODS: It was studied with orthogonal design to analysis three factors of inclusion rate such as the weight ratio between HDCA and beta-Cyclodextrin, the temperature and the reaction time. Fourier transform infrared spectroscopy, scanning electron microscopy, taste and solubility test were used to identify the inclusion compound. RESULTS: Stable inclusion compound was made by HDCA and beta-Cyclodextrin. The weight ratio between HDCA and beta-Cyclodextrin was the most important factor. The alpha-Hydrocycholic-beta-Cyclodextrin inclusion compound taste and the solubility were improved. CONCLUSION: The optimum preparation condition is alpha-Hydrocycholic : beta-Cyclodextrin = 8.67 : 1, the inclusion temperature is 60 degrees C and the inclusion time is 30 minutes.