Recent efforts in understanding the epitranscriptome have shown that a diverse set of modifications to RNA represent a new pervasive mechanism of gene regulation, with roles in stem cell homeostasis and disease. N6-methyladenosine (m6A) is an evolutionarily conserved RNA modification and one of the most abundant found on polyadenylated RNA [1, 2]. The modification is predominantly deposited on mRNA by the METTL3/METTL14 methyltransferase complex [3, 4]. The majority of the reported phenotypes connected to METTL3/METTL14 function have so far utilised genetic knock-down or knock-out approaches which have been proven fairly pleiotropic, mainly due to the significant negative impact on the general m6A complex [3, 4]. Lack of reagents and strategies to selectively block the catalytic activity of METTL3 without affecting any of its other functions and interactions has hindered investigation of catalysis-specific METTL3 activity. We recently showed that pharmacological inhibition of the catalytic activity of METTL3, using the first-inclass small molecule STM2457, is a novel therapeutic strategy against acute myeloid leukaemia (AML)[5]. While no toxicity or long-term effects on normal blood counts were observed after in vivo pharmacological inhibition using STM2457, the potential impact of the isolated catalytic inhibition of METTL3 on normal haematopoiesis remained elusive. To address this, here we utilize a high-resolution single cell RNA sequencing (scRNA-seq) approach to understand: 1) the effect of catalytic inhibition of METTL3 on different lineages within normal haematopoiesis and 2) its specific impact on haematopoietic stem cell fate decisions in vivo.

To investigate the above, we initially performed in vivo studies using wild-type CB57BL/6 N mice treated daily with either vehicle or 50 mg/kg of STM2457 over the course of 2 weeks (Fig. S1A). We confirmed effective and selective in vivo catalytic inhibition of METTL3 as total m6A modification levels on RNA were significantly reduced after treatment with STM2457 while no changes were detected on m6 2A RNA modification levels (Fig. 1 A). Consistent with previous reports using METTL3 knock-out (KO) mouse models