Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. NSun2 knockout mouse models and Klf2 cells obtained from individuals with Dubowitz-like syndrome, we show that cytosine-5 tRNA methylation is a very common modification and is required to mediate cellular survival during stress responses. We reveal that fragmentation of tRNAs into short non-coding RNAs is perturbed in mouse and patient cells lacking the NSun2 protein, and identify aberrant accumulation of cleaved tRNAs as one mechanism by which mutations in a RNA methyltransferase can result in neurological abnormalities in mice and humans. Results Cytosine-5 methylation is a common modification in actively transcribed tRNAs Recent high-throughput RNA methylation profiling identified tRNAs as the most common cytosine-5 methylated RNA species (Squires and (Supplementary Fig S4A and B), and tRNA cleavage also occurred in wild-type NSun2-expressing cells in response to oxidative stress (NaAsO2) (Supplementary Fig S4C). Therefore, we speculated that inhibition of NSun2 rather occurred on protein level. To investigate how the enzymatic activity of NSun2 might be inhibited in response to stress, we examined the cellular localisation of NSun2 in primary human and mouse skin cultures exposed to UVB radiation. The vast majority of the NSun2 protein is found in the nucleoli, where tRNA methylation takes place (Fig?(Fig3A;3A; untreated; arrow) (Colonna & Kerr, 1980; Frye & Watt, 2006; Hussain (Supplementary Fig S4I). After 24?h of UV treatment, the number of apoptotic cells was higher in NSun2-depleted skin (Fig?(Fig3J).3J). To show that cellular survival after stress directly depended on NSun2 methyltransferase activity, we measured the percentage of dead cells in response to UV radiation in primary human keratinocytes overexpressing either wild-type or a mutant NSun2 construct (K190M), that is unable to methylate tRNA (Hussain synthesised tRNAs are not methylated at C38 and therefore can still be m5C-methylated at C38 in the presence of Dnmt2. We then incubated the synthetic tRNAs with cell 1223001-51-1 supplier lysates from NSun2+/? (NSun2-expressing cells) (Fig?(Fig6A;6A; NSun2) or NSun2?/? human fibroblasts (NSun2 lacking cells) (Fig?(Fig6A;6A; no NSun2). After incubation with cell lysates, we measured the ratio of purified cleaved 5 ends versus full-length tRNA (Fig?(Fig6A6A and B, and Supplementary Fig S8B). Enrichment of 5 1223001-51-1 supplier tRNA fragments correlated with loss of methylation at C48/49 (Fig?(Fig6B;6B; left hand panel; Supplementary Fig S8BCD). We confirmed increased cleavage of non-methylated overmethylated tRNA after incubation with NSun2?/? lysates (Fig?(Fig6B;6B; right hand panel; Supplementary Fig S8E). To further determine that m5C-methylation at C48/49 shielded tRNA from angiogenin binding, we analysed the amount of angiogenin bound to the purified tRNA. Although the quantities of full-length C48/49G and non-methylated tRNAs were reduced (Fig?(Fig6B),6B), the constructs bound angiogenin with higher affinity (Fig?(Fig6C,6C, and Supplementary Fig S8F), indicating that methylated C48/49 protected the tRNA from binding to and being cleaved by angiogenin. We obtained the same results using methylated and non-methylated tRNA LysCTT (Supplementary Fig S8G and H). tRNA LysCTT is not a Dnmt2 substrate. Figure 6 Cytosine-5 methylation protects from cleavage by angiogenin and angiogenin inhibition rescues elevated stress levels of NSun2?/? cells 5 tRNAs-induced stress can be rescued by inhibition of angiogenin To test whether cleavage of tRNAs lacking m5C at position 48 and 49 was solely dependent on angiogenin or included additional endonucleases, we inhibited angiogenin by RNAi and the small-molecule inhibitor “type”:”entrez-nucleotide”,”attrs”:”text”:”N65828″,”term_id”:”1217454″,”term_text”:”N65828″N65828 (Kao and causing a syndromic disorder characterised by growth and neuro-developmental deficiencies in mice and human. Both inhibition of tRNA cleavage and oxidative stress pathways during mouse embryogenesis abrogate the stress-induced pathways and revert cell size and survival to normal. Thus, we identified the first mechanistic link between loss-of-function of the gene and the neuro-developmental disorder in humans. tRNA and tRNA modifications in human disease tRNAs are a fundamental component of the translation 1223001-51-1 supplier machinery and genetic mutations that affect mRNA translation commonly contribute to human cognitive and behavioural diseases (Scheper (Schaefer and hybridisation (FISH) Co-localisation of NSun2 with stress granules or processing bodies was performed as reported (Kedersha & Anderson, 2007). Briefly, cells were fixed for 15?min with 4% paraformaldehyde.
Alphaviruses are a group of widely distributed human and animal pathogens. of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 conversation with alphaviruses to develop new attenuated variations of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates. Author Summary Alphaviruses represent a group of highly important human pathogens, which are transmitted by mosquito vectors between vertebrate hosts. Alphavirus replication in vertebrates depends on their ability to interfere with host antiviral responses on both cellular and organismal levels. The recognition of cellular factors, which impact computer virus replication, and characterization of their functions may show crucial for the design of new effective vaccine candidates. We have exhibited that the protein product of one of the interferon-stimulated genes, IFIT1, is usually a potent inhibitor of translation of the incoming alphavirus genomes and ultimately, computer virus replication. The secondary structure of the 5untranslated regions (5UTRs) of alphavirus genomes was shown to play a crucial role in alphavirus resistance to this inhibitory effect. Moreover, in IFIT1-conveying cells, wt alphaviruses exhibiting low sensitivity to IFIT1 also were found to induce high levels of type I IFN. Altogether, our data show that alphavirus 5UTRs were evolutionarily selected to meet the requirements of both functioning as promoters for positive- and negative-strand RNA synthesis and supporting the resistance to inhibitory effects of IFIT1. We further exploited this new knowledge to develop mutated alphaviruses, which displayed higher sensitivity to IFIT1 and more attenuated phenotypes. Introduction The Alphavirus genus Valrubicin supplier in the family contains 30 species Klf2 and has a worldwide distribution . Most alphaviruses are transmitted by mosquito vectors between amplifying vertebrate hosts . In vertebrates, alphaviruses cause acute infections, characterized by high titer viremia that allows contamination of mosquitoes during blood feeding. A number of alphaviruses, including Venezuelan (VEEV) and eastern (EEEV) equine encephalitis viruses, and chikungunya computer virus (CHIKV), are globally important, emerging public health threats. These viruses can cause epidemics of severe meningoencephalitis with frequent lethal outcomes, or polyarthritis with excruciating and chronic joint pain . Over the last ten years, there have been multiple outbreaks of CHIKV contamination with hundreds of thousands Valrubicin supplier of people infected [4C7], including an ongoing epidemic in the Americas with more than 40 countries affected and over 1.1 million suspected cases. Epizootic stresses of VEEV and EEEV are almost universally lethal for equids . In addition, the second option viruses can be transmitted efficiently by aerosol , are highly stable in lyophilized form, and were developed previously as biological warfare brokers . In spite of their public health threat, the pathogenesis of alphaviruses on the molecular and cellular levels remains poorly comprehended, and no approved vaccines or therapies exist for any of them. Alphavirus attacks are delicate Valrubicin supplier to type I IFN both and [10C12]. Rodents faulty in IFN signaling succumb to most alphavirus attacks within a few times . IFN treatment induce a huge established of IFN-stimulated genetics (ISGs), whose proteins items prevent infections with many pathogens, including alphaviruses [14C17]. Although hundreds of ISGs possess been referred to, just few possess got their antiviral features described unambiguously, for alphaviruses [18C24] particularly. The participation of therefore many mobile genetics suggests that the antiviral response might Valrubicin supplier end up being extremely unnecessary against a provided virus. Such redundancy would decrease the likelihood of selection of pathogen mutants resistant to an ISG item. Nevertheless, it is certainly also possible that just a subset of ISGs is certainly important for security against a particular virus or groupings of related pathogens. Hence, id of pathogen-specific ISGs might business lead to advancement of targeted therapeutics lacking.