Thermo-sensing mechanisms of splicing control by nuclear stress bodies

Nuclear stress bodies (nSBs) are stress-inducible membraneless organelles formed on HSATIII long noncoding RNAs (lncRNAs) that regulate pre-mRNA splicing during thermal stress recovery. During stress, dephosphorylated serine/arginine-rich splicing factors (SRSFs) accumulate in nSBs, whereas upon stress removal, their kinase CLK1 is recruited to rephosphorylate SRSFs, thereby promoting target intron detention. However, the mechanism underlying CLK1 localization to nSBs has remained unclear. Using HeLa cells and cell-free reconstitution, we identify CLK1 Ser341 phosphorylation as a critical determinant of its nSB localization and define its regulatory mechanism. Ser341 is phosphorylated under normal conditions, dephosphorylated by protein phosphatase 1(PP1) during stress, and rephosphorylated by RIOK2 during recovery, thus enabling CLK1 localization to nSBs specifically during recovery. We further identify PPP1R2, an entirely intrinsically disordered PP1 inhibitory subunit, as a reversible thermosensor that dissociates under stress to activate PP1. Together, our findings reveal multilayered thermosensing mechanisms that coordinate the staged localization of SRSFs and CLK1 to nSBs, thereby regulating temperature-dependent pre-mRNA splicing.