Loss of 5-methylcytosine alters the biogenesis of vault-derived small RNAs to coordinate epidermal differentiation.

The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.We thank everybody who provided us with reagents, in particular we thank James Stevenin for sending us recombinant SRSF2. We gratefully acknowledge the support of all the WT-MRC Stem Cell Institute core facility managers. This work was funded by Cancer Research UK (CR-630 UK) and the European Research Council (ERC). Parts of this research in Michaela Frye's laboratory was supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. Juri Rappsilber’s laboratory was supported by Wellcome Trust Senior Research Fellowship (084229). Gracjan Michlewski’s laboratory was supported by the MRC Career Development Award (G10000564), Wellcome Trust Seed Award (210144/Z/18/Z) and Wellcome Trust Centre for Cell Biology Core Grants (077707 and 092076). Abdulrahim Sajini was supported by a scholarship from the University of Tabuk and Khalifa University of Science and Technology Faculty start-up award number FSU-2018-01. Rebecca Wagner was supported by the Wellcome Trust PhD Programme in Stem Cell Biology & Medicine

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.This work was funded by a Cancer Research UK Senior Fellowship (C10701/A15181), the European Research Council (ERC; 310360), and the Medical Research Council UK (MR/M01939X/1). Part of this work was carried out in the framework of the European COST action EPITRAN 16120. SK receives funding from the DFG (KE1943/3-1). The funders had no role in study design, data collection, and analysis, decision to publish, 5 or preparation of the manuscript

Stem cell function and stress response are controlled by protein synthesis.

Whether protein synthesis and cellular stress response pathways interact to control stem cell function is currently unknown. Here we show that mouse skin stem cells synthesize less protein than their immediate progenitors in vivo, even when forced to proliferate. Our analyses reveal that activation of stress response pathways drives both a global reduction of protein synthesis and altered translational programmes that together promote stem cell functions and tumorigenesis. Mechanistically, we show that inhibition of post-transcriptional cytosine-5 methylation locks tumour-initiating cells in this distinct translational inhibition programme. Paradoxically, this inhibition renders stem cells hypersensitive to cytotoxic stress, as tumour regeneration after treatment with 5-fluorouracil is blocked. Thus, stem cells must revoke translation inhibition pathways to regenerate a tissue or tumour.This work was funded by Cancer Research UK (CR-UK), Worldwide Cancer Research, the Medical Research Council (MRC), the European Research Council (ERC), and EMBO. Research in Michaela Frye's laboratory is supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Cambridge Stem Cell Institute.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1828

FTO m6A Demethylase in Obesity and Cancer: Implications and Underlying Molecular Mechanisms

Fat mass and obesity-associated protein (FTO) is the first reported RNA N6-methyladenosine (m6A) demethylase in eukaryotic cells. m6A is considered as the most abundant mRNA internal modification, which modulates several cellular processes including alternative splicing, stability, and expression. Genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) within FTO to be associated with obesity, as well as cancer including endometrial cancer, breast cancer, pancreatic cancer, and melanoma. Since the initial classification of FTO as an m6A demethylase, various studies started to unravel a connection between FTO’s demethylase activity and the susceptibility to obesity on the molecular level. FTO was found to facilitate adipogenesis, by regulating adipogenic pathways and inducing pre-adipocyte differentiation. FTO has also been investigated in tumorigenesis, where emerging studies suggest m6A and FTO levels are dysregulated in various cancers, including acute myeloid leukemia (AML), glioblastoma, cervical squamous cell carcinoma (CSCC), breast cancer, and melanoma. Here we review the molecular bases of m6A in tumorigenesis and adipogenesis while highlighting the controversial role of FTO in obesity. We provide recent findings confirming FTO’s causative link to obesity and discuss novel approaches using RNA demethylase inhibitors as targeted oncotherapies. Our review aims to confirm m6A demethylation as a risk factor in obesity and provoke new research in FTO and human disorders

FTO m6A Demethylase in Obesity and Cancer: Implications and Underlying Molecular Mechanisms

Fat mass and obesity-associated protein (FTO) is the first reported RNA N6-methyladenosine (m6A) demethylase in eukaryotic cells. m6A is considered as the most abundant mRNA internal modification, which modulates several cellular processes including alternative splicing, stability, and expression. Genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) within FTO to be associated with obesity, as well as cancer including endometrial cancer, breast cancer, pancreatic cancer, and melanoma. Since the initial classification of FTO as an m6A demethylase, various studies started to unravel a connection between FTO’s demethylase activity and the susceptibility to obesity on the molecular level. FTO was found to facilitate adipogenesis, by regulating adipogenic pathways and inducing pre-adipocyte differentiation. FTO has also been investigated in tumorigenesis, where emerging studies suggest m6A and FTO levels are dysregulated in various cancers, including acute myeloid leukemia (AML), glioblastoma, cervical squamous cell carcinoma (CSCC), breast cancer, and melanoma. Here we review the molecular bases of m6A in tumorigenesis and adipogenesis while highlighting the controversial role of FTO in obesity. We provide recent findings confirming FTO’s causative link to obesity and discuss novel approaches using RNA demethylase inhibitors as targeted oncotherapies. Our review aims to confirm m6A demethylation as a risk factor in obesity and provoke new research in FTO and human disorders

Synthesis, Characterization, Biological Evaluation and DNA Interaction Studies of 4-Aminophenol Derivatives: Theoretical and Experimental Approach

In the present study, five 4-aminophenol derivatives (4-chloro-2-(((4-hydroxyphenyl)imino)methyl)phenol(S-1), 4-((4-(dimethylamino)benzylidene)amino)phenol(S-2), 4-((3-nitrobenzylidene)amino)phenol(S-3), 4-((thiophen-2-ylmethylene)amino)phenol(S-4) and 4-(((E)-3-phenylallylidene)amino)phenol(S-5)) were synthesized and characterized by FT-IR, 1H-NMR, 13C-NMR and elemental analyses. The synthesized compounds were tested for their antimicrobial (Gram-positive and Gram-negative bacteria and Saccharomyces cervesea fungus) and antidiabetic (α-amylase and α-glucosidase inhibitory) activities. All the compounds showed broad-spectrum activities against the Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis sub. sp spizizenii (ATCC 6633), Bordetella bronchiseptica (ATCC 4617) and Saccharomyces cerevisiae (ATCC 9763) strains. The newly synthesized compounds showed a significant inhibition of amylase (93.2%) and glucosidase (73.7%) in a concentration-dependent manner. Interaction studies of Human DNA with the synthesized Schiff bases were also performed. The spectral bands of S-1, S-2, S-3 and S-5 all showed hyperchromism, whereas the spectral band of S-4 showed a hypochromic effect. Moreover, the spectral bands of the S-2, S-3 and S-4 compounds were also found to exhibit a bathochromic shift (red shift). The present studies delineate broad-spectrum antimicrobial and antidiabetic activities of the synthesized compounds. Additionally, DNA interaction studies highlight the potential of synthetic compounds as anticancer agents. The DNA interaction studies, as well as the antidiabetic activities articulated by the molecular docking methods, showed the promising aspects of synthetic compounds

Colorimetric Detection of Organophosphate Pesticides Based on Acetylcholinesterase and Cysteamine Capped Gold Nanoparticles as Nanozyme

Organophosphates (OPs) are neurotoxic agents also used as pesticides that can permanently block the active site of the acetylcholinesterase (AChE). A robust and sensitive detection system of OPs utilising the enzyme mimic potential of the cysteamine capped gold nanoparticles (C-AuNPs) was developed. The detection assay was performed by stepwise addition of AChE, parathion ethyl (PE)-a candidate OP, acetylcholine chloride (ACh), C-AuNPs, and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the buffer solution. The whole sensing protocol completes in 30–40 min, including both incubations. The Transmission Electron Microscopy (TEM) results indicated that the NPs are spherical and have an average size of 13.24 nm. The monomers of C-AuNPs exhibited intense catalytic activity (nanozyme) for the oxidization of TMB, revealed by the production of instant blue colour and confirmed by a sharp peak at 652 nm. The proposed biosensor’s detection limit and linear ranges were 5.8 ng·mL−1 and 11.6–92.8 ng·mL−1, respectively, for PE. The results strongly advocate that the suggested facile colorimetric biosensor may provide an excellent platform for on-site monitoring of OPs

Comparative study of microscale and macroscale technique for encapsulation of Calotropis gigantea extract in metal-conjugated nanomatrices for invasive ductal carcinoma

Abstract The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV–Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.This work was funded by a Cancer Research UK Senior Fellowship (C10701/A15181), the European Research Council (ERC; 310360), and the Medical Research Council UK (MR/M01939X/1). Part of this work was carried out in the framework of the European COST action EPITRAN 16120. SK receives funding from the DFG (KE1943/3-1). The funders had no role in study design, data collection, and analysis, decision to publish, 5 or preparation of the manuscript

The Potential Use of Mesenchymal Stem Cells and Their Derived Exosomes as Immunomodulatory Agents for COVID-19 Patients

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing lethal acute respiratory disease emerged in December 2019. The World Health Organization named this disease “COVID-19” and declared it a pandemic on March 11, 2020. Many studies have shown that mesenchymal stem cells (MSCs) and their exosomes (MSCs-Exo), which are isolated from allogenic bone marrow stem cells, significantly lower the risk of alveolar inflammation and other pathological conditions associated with distinct lung injuries. For example, in acute respiratory distress syndrome (ARDS) and pneumonia patients, MSCs-Exo and MSCs provide similar healing properties and some clinical trials have used cell-based inhalation therapy which show great promise. MSCs and MSCs-Exo have shown potential in clinical trials as a therapeutic tool for severely affected COVID-19 patients when compared to other cell-based therapies, which may face challenges like the cells’ sticking to the respiratory tract epithelia during administration. However, the use of MSCs or MSCs-Exo for treating COVID-19 should strictly adhere to the appropriate manufacturing practices, quality control measurements, preclinical safety and efficacy data, and the proper ethical regulations. This review highlights the available clinical trials that support the therapeutic potential of MSCs or MSCs-Exo in severely affected COVID-19 patients