Xist recruits the silencing protein SPEN through a mechanism in which SR splicing factors, particularly SRSF1, bind to GC-rich motifs in Repeat A and serve as bridging adaptors that enable SPEN's N-terminus to recognize SR-protein-rich assemblies, thereby coordinating interactions with the m6A methylation, RNA turnover, and transcriptional elongation machineries to induce gene silencing—a general regulatory mechanism that SPEN also employs genome-wide to repress autosomal genes bearing similar SR-protein-enriched RNA features.

The lncRNA Xist represents a paradigm to understand the mechanisms of RNA-mediated gene silencing in mammals, which remain largely unresolved. To induce silencing, Xist recruits the RNA-binding protein SPEN through its 5′-proximal Repeat A domain. Yet, how Repeat A recruits SPEN and how SPEN coordinates silencing remain unclear. We report that sequences in Repeat A critical for SPEN recruitment directly bind SR-rich splicing factors. SRSF1, one such factor, is required for optimal SPEN recruitment and its RS-domain recruits SPEN when tethered to Xist. SPEN and SR-protein-binding motifs promote Repeat A’s association with many proteins, including the m6A machinery and elongating RNA polymerase II. SPEN also represses autosomal genes where its recruitment coincides with SR-protein binding. Our results reveal an unexpectedly essential role for splicing factors in coordinating silencing by Xist and suggest that the sensing of SR-protein-rich assemblies is a general mechanism through which SPEN targets genes for repression.