Multivalent Scaffolding by XIST E-Repeats Tunes RNA Binding Protein Cooperativity and Lowers the Phase Separation Threshold

Abstract

The long-noncoding RNA XIST coordinates the random silencing of one X-chromosome in female placental mammals as a class of chromosomal dosage compensation across sexes. Critical to this process is the diffusion of XIST foci across the 155 Mb of the X-chromosome to achieve 96% repression. The E-Repeat domain on XIST primarily drives this process through the recruitment of several RNA-binding proteins, which seed condensate formation for controlled distal spreading. To date, there is a lack of biophysical characterization of interactions between the E-Repeats and its protein binding partners. Herein, we define the interaction, in vitro, between E-Repeat RNA and proteins PTBP1, ELAVL1, TDP43, and hnRNPU by applying a combination of bioinformatics, in silico characterization, and biochemical assays. We designed minimal E-Repeat RNA constructs to study the molecular mechanisms of RBP recognition and the E-Repeat ensemble. We find that all proteins sustain nanomolar interactions with single E-Repeat RNA. Through a systematic domain study, we deduce protein domain-specific RNA binding affinities, providing insight into mechanisms of E-Repeat binding. With four E-Repeats, we find that PTBP1 and ELAVL1 form higher-order assemblies and demonstrate protein competition and co-occupancy, reflecting previous in cellulo CLIP experiments. Finally, we find that PTBP1 and ELAVL1 phase separate with the four-repeat minimal constructs under minimal in vitro conditions, recapitulating the cellular behavior crucial to XIST function. All together, our findings provide mechanistic insight into the amplified impact of XIST, which at approximately 1 molecule per megabase can modulate long-range chromatin organization, illuminating another aspect of lncRNA potency, and potentially informing engineered derivatives of XIST and other lncRNAs as genetic therapeutics.