Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.

Automated summary

The study reveals that Xist RNA directs X chromosome inactivation in mammals by spreading along the chromosome and recruiting chromatin modifiers, with a feedback mechanism linking Xist RNA synthesis and degradation, as well as physical coupling between preceding and newly synthesized Xist RNA molecules. SPEN, a key factor for Xist-mediated gene silencing, plays a distinct role in Xist RNA localization, stability, and coupling behaviors.