The N6-methyladenosine (m 6 A) is an abundant internal RNA modification 1,2 catalysed predominantly by the METTL3-METTL14 methyltransferase complex 3,4 . The m 6 A writer METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but its true therapeutic importance is still unknown [5][6][7] . Here we present the identification and characterisation of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) and its co-crystal structure bound to METTL3/METTL14. Treatment with (Extended Data Fig. 2f). These data demonstrate that STM2457 is a highly potent, specific and bioavailable inhibitor of METTL3, suitable for in vivo investigations. Cellular and molecular effects of STM2457To study the anti-leukaemic potential of STM2457 we examined the proliferation of a panel of human AML cell lines post-treatment and detected significant growth reduction in a concentration-dependent manner (Fig. 2a) while we found that STM2457 did not affect the colony-forming ability of normal human cord blood CD34 + cells (Extended Data Fig. 3a). We also observed no impact on the proliferation of MOLM-13 cells treated with the control small molecule STM2120, unlike our observations with STM2457 (Extended Data Fig. 3b). Additionally, treatment with STM2457 significantly reduced the clonogenic potential of primary murine AML cells (Fig. 2b and Extended Data Fig. 3c), while having no effect on normal haematopoietic stem and progenitor cells (HSPCs) (Fig. 2c). Pharmacological inhibition of METTL3 also caused significant myeloid differentiation 6,11 and cell cycle arrest in MOLM-13 and primary murine AML cells (Fig. 2d, e). In contrast, the same effects were not identified using the non-leukaemic haemopoietic cell line HPC7 (Fig. 3e and Extended Data Fig. 3d). Moreover, treatment with STM2457 induced apoptosis in human and mouse AML models but not in normal non-leukaemic haemopoietic cells (Fig. 2f and Extended Data Fig. 3e). To assess the impact of pharmacological inhibition of METTL3 on two known METTL3 biomarkers associated with AML, SP1 6,12 and BRD4 13,14 , we treated MOLM-13 cells with STM2457 and observed a dose-dependent reduction of SP1 and BRD4 protein levels (Fig. 2g). Notably, ectopic expression of SP1 significantly reduced the sensitivity of MOLM-13 cells to STM2457 (Extended Data Fig. 3f, g). These data establish that the catalytic function of METTL3 is important for leukaemia growth, in line with previous findings 6,7,15 . We next sought to investigate the molecular mechanism by which STM2457 affects AML. RNAseq analysis of MOLM-13 cells treated with STM2457 revealed 1,338 up-regulated and 489 down-regulated genes (Extended Data Fig. 4a and Supplementary Table 1). Gene ontology (GO) analysis of the differentially expressed genes showed enrichment in pathways related to myeloid differentiation, cell cycle and leukaemia progression (Extended Data Fig. 4b, c) in close agreement with our phenotypic observations (Supplementary Table 2). To examine the impact of the pharmac...

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B, and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.