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RNA 5-methylcytosine (m 5 C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m 5 C in UM. Initially, we identified significantly elevated global RNA m 5 C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m 5 C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m 5 C-RIP-qPCR, and luciferase assay identified β-Catenin (CTNNB1) as a direct target of NSUN2-mediated m 5 C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m 5 C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.

Loss-of-function mutations in the cytosine-5 RNA methylase NSUN2 cause neurodevelopmental disorders in humans, yet the underlying cellular processes leading to the symptoms that include microcephaly remain unclear. Here, we show that NSUN2 is expressed in early neuroepithelial progenitors of the developing human brain, and its expression is gradually reduced during differentiation of human neuroepithelial stem (NES) cells in vitro. In the developing Nsun2 -/- mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in Nsun2 -/- brains. Neural differentiation of NES cells is impaired by both NSUN2 depletion and the presence of angiogenin. Since repression of NSUN2 also inhibited neural cell migration toward the chemoattractant fibroblast growth factor 2, we conclude that the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing technologies have enabled the transcriptome to be profiled at a previously unprecedented speed and depth. This yielded insights into fundamental transcriptomic processes such as gene transcription, RNA processing, and mRNA splicing. Immunoprecipitation-based transcriptomic methods such as individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) have also allowed high-resolution analysis of the RNA interactions of a protein of interest, thus revealing new regulatory mechanisms. We and others have recently modified this method to profile RNA methylation, and we refer to this customized technique as methylation-iCLIP (miCLIP). Variants of miCLIP have been used to map the methyl-5-cytosine (m5C) or methyl-6-adenosine (m6A) modification at nucleotide resolution in the human transcriptome. Here we describe the m5C-miCLIP protocol, discuss how it yields the nucleotide-resolution RNA modification maps, and comment on how these have contributed to the new field of molecular genetics research coined "epitranscriptomics."

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy. [ABSTRACT FROM AUTHOR]

5‐Methylcytosine (m5C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1‐7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell‐active inhibitors of these enzymes are lacking. Here, we use cysteine‐directed activity‐based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross‐reactivity with other human NSUNs and exhibit good proteome‐wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2‐tRNA interactions in cancer cells, leading to a global reduction in tRNA m5C content. Our findings thus highlight the potential to create isotype‐selective and cell‐active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine. [ABSTRACT FROM AUTHOR]

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the role of NSUN2 (NOP2/Sun RNA Methyltransferase Family Member 2) in hepatocellular carcinoma (HCC). The expressions of NSUN2 and FZR1 were measured. Cell viability, proliferation, and apoptosis were assessed. HCC xenograft in nude mouse model was established. Tumor weight and volume were examined. Tumor tissues were collected for immunohistochemistry (IHC). TCGA database analysis and clinical sample testing suggested that the transcript levels of NSUN2 and FZR1 were increased in HCC tissues. NSUN2 knockdown inhibited HCC cell viability and proliferation, and promoted cell apoptosis. Moreover, the effects of NSUN2 could be countered by overexpressing FZR1. In animal experiment, NSUN2 silencing suppressed tumor growth in nude mice by downregulating FZR1. In conclusion, NSUN2 has a regulatory effect on HCC cell proliferation and apoptosis. NSUN2 knockout could inhibit cellular processes in HCC and tumor growth, likely via FZR1 inhibition. This finding has not only revealed the role of NSUN2 in HCC growth, but also suggests a promising target for HCC treatment. [ABSTRACT FROM AUTHOR]