Developmental Xist induction is mediated by enhanced splicing

X-inactive-specific transcript (Xist) is a long noncoding RNA (lncRNA) essential for inactivating one of the two X chromosomes in mammalian females. Random X chromosome inactivation is mediated by Xist RNA expressed from the inactive X chromosome. We found that Xist RNA is unspliced in naïve embryonic stem (ES) cells. Upon differentiation, Xist splicing becomes efficient across all exons independent of transcription, suggesting interdependent or coordinated removal of Xist introns. In female cells with mutated polypyrimidine tract binding protein 1 (Ptbp1), differentiation fails to substantially upregulate mature Xist RNA because of a defect in Xist splicing. We further found both Xist(129) and Xist(CAS) RNA are unspliced in Mus musculus 129SvJ/Mus castaneous (CAS) hybrid female ES cells. Upon differentiation, Xist(129) exhibits a higher splicing efficiency than Xist(CAS), likely contributing to preferential inhibition of the X(129) chromosome. Single cell analysis shows that the allelic choice of Xist splicing is linked to the inactive X chromosome. We conclude post-transcriptional control of Xist RNA splicing is an essential regulatory step of Xist induction. Our studies shed light on the developmental roles of splicing for nuclear-retained Xist lncRNA and suggest inefficient Xist splicing is an additional fail-safe mechanism to prevent Xist activity in ES cells.