Impaired stem cell differentiation and somatic cell reprogramming in DIDO3 mutants with altered RNA processing and increased R-loop levels

Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are biological processes governed by antagonistic expression or repression of a largely common set of genes. Accurate regulation of gene expression is thus essential for both processes, and alterations in RNA processing are predicted to negatively affect both. We show that truncation of the DIDO gene alters RNA splicing and transcription termination in ESC and mouse embryo fibroblasts (MEF), which affects genes involved in both differentiation and reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify the underlying molecular mechanism. We found that DIDO3 interacts with the helicase DHX9, which is involved in R-loop processing and transcription termination, and that DIDO3-exon16 deletion increases nuclear R-loop content and causes DNA replication stress. Overall, these defects result in failure of ESC to differentiate and of MEF to be reprogrammed. MEF immortalization restored impaired reprogramming capacity. We conclude that DIDO3 has essential functions in ESC differentiation and somatic cell reprogramming by supporting accurate RNA metabolism, with its exon16-encoded domain playing the main role.

胚胎干细胞（ESC）分化与体细胞重编程是由一大批共有基因的拮抗表达或抑制所调控的生物学过程。因此，基因表达的精准调控对这两个过程均至关重要，而RNA加工异常据推测会对二者产生负面影响。我们的研究表明，DIDO基因的截短会改变ESC与小鼠胚胎成纤维细胞（MEF）中的RNA剪接与转录终止过程，进而影响分化与重编程相关基因的表达。我们结合转录组学、蛋白质相互作用及细胞实验来解析其潜在分子机制。研究发现，DIDO3与参与R环加工及转录终止的解旋酶DHX9存在相互作用；而DIDO3外显子16的缺失会增加细胞核内R环含量，并引发DNA复制应激。总体而言，这些缺陷会导致ESC无法完成分化，以及MEF无法被重编程。MEF永生化可恢复受损的重编程能力。综上，我们认为DIDO3通过维持精准的RNA代谢过程，在ESC分化与体细胞重编程中发挥必需功能，其由外显子16编码的结构域在此过程中起到主要作用。