Phosphorylation-dependent Charge Blocks Regulate the Coarsening of Nuclear Speckle Networks

Nuclear speckles (NSs) are viscoelastic network fluids formed via phase separation coupled to percolation (PSCP). Intermolecular cross-links of SRRM2, a key scaffold of NSs lead to the emergence of system-spanning networks, although the physiochemical grammar governing SRRM2 PSCP remains poorly decoded. Here, we demonstrate that SRRM2 is extensively phosphorylated within the intrinsically disordered domain (IDR) that are prominently positively charged. Rather than attaching phosphate groups to specific sites, the purpose of phosphorylation is to create alternating charge blocks. Employing a suite of biophysical approach, we show that in contrast to the leading theories that charge blocks lower condensation threshold of biopolymers, this specific charge pattern does not markedly alter the percolation threshold of SRRM2 in cells. Instead, SRRM2 charge blocks intensify intra-network molecular interactions to modulate the material properties of mesoscopic SRRM2 condensates. We further identify casein kinase 2 (CK2) as the upstream enzyme to catalyze SRRM2 phosphorylation. Phosphorylation of SRRM2 IDR by CK2 facilitates NS relaxation under physiological condition, but can be employed to safeguard genome stability in times of DNA damage. Our findings reveal important regulatory mechanisms of charge blocks and functionality in modulating the material properties of biomolecular condensates in cells. We interpretate our observation in the theoretical framework of stickers-and-spacers model. In addition to the chemistry- or sequence-specific associative interaction, spacers (e.g. SRRM2 IDR) can also engage in charge pattern-specific molecular interactions, which are spatiotemporally regulated, to dictate the biophysical properties of percolated macromolecular assemblies. Stable cell lines were generated by co-transfection of Piggybac transposon vector and Piggybac transposase (Supper Piggybac Transposase). After 48h transfection, clonal populations of each cell line were obtained by flow cytometric cell sorting. Established cell lines were grown as described above in medium.RNA seq were used to detecte the splicing changes.The splicing differences of piggybac-WT, piggybac-SA and piggybac-SD cell lines were compared.