DIDO3-specific exon16 regulates RNA metabolism and is necessary for differentiation and reprogramming

Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are similar processes, governed by transcription factor networks whose expression is controlled by precise RNA metabolism. Here we demonstrate that, following conditional deletion of C terminal-exon16, ESC are able to self-renew but their in vitro differentiation is compromised, and MEF (murine embryonic fibroblasts) show impaired somatic cell reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify a DIDO3 regulatory network in ESC, and show that exon16 deletion modulates RNA splicing and termination (3UTR read-through), R loops, and replication stress. DIDO3-exon16 interacts with the helicase Dhx9, which processes R loops; its deletion leads to increased R-loop formation. These effects, specific to the DIDO3dE16 mutant, are not observed in cells with DIDO N-terminal truncation. DIDO3dE16 MEF reprogramming is rescued by cell immortalization. Finally, we reassessed the hypothesis that DIDO3-exon16 regulates RNA metabolism and is necessary for ESC differentiation and somatic cell reprogramming.