SRA

Upon?stimulation by extrinsic stimuli,?stem cells initiate a program that enables differentiation or self-renewal.?Disruption?of?the stem-state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem-state switch are known, major regulatory mechanisms remain unclear. Here, we show this switch involves a global increase in splicing efficiency coordinated by DNMT3A, an enzyme typically involved in DNA methylation. Proper activation?of murine and human embryonic and hematopoietic stem cells depends on mRNA processing influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation?function?of DNMT3A is not required?and loss?of DNMT3A leads to?impaired?splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukemias. Together,?our results reveal a modality through which DNMT3A?and?the spliceosome?govern exit from the stem-state towards differentiation. Overall design: mRNA sequencing (RNA-seq) of DNMT3A WT and KO cells in mouse and human HSCs and ESCs Total RNA was isolated from mouse HSCs and human HSPCs using PicoPure RNA isolation kit (KIT0204). RNA from mESC and hESC was isolated using the Qiagen mini kit. Quality of isolated RNA was verified with Nanodrop and tapestation prior to library preparation. 500-750ng of total RNA was used was used as input for the Illumina TruSeq Stranded mRNA LT Prep Kit (20020594). Libraries were made following Illumina's recommended protocol. Amplified libraries were purified and quantified using the KAPA quantification kit. RNA sequencing libraries were sequenced on an Illumina NextSeq 500 instrument (paired-end).