Condensation of ZFP207 and U1 snRNP promotes spliceosome assembly [RAP-seq]

U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 largely recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs) to forms biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring proper U1 snRNP function and efficient spliceosome assembly. Briefly, harvest the ZFP207 AID mESCs with or without IAA treatment and incubate them with 0.5 mg/ml AMT solution on ice for 15 minutes. Transfer the cell suspension to a 6 cm dish and irradiate with 365 nm UV for 15 minutes. Collect the cells, wash with PBS, and spin down. Then add nuclear lysis buffer, and sonicate the samples at 30% amplitude for 5 minutes. Biotinylated U1 probes were added and hybridize at 39°C for 1 hour, then add streptavidin beads and incubate at 39°C for another 1 hour. After thorough wash with 0.2×SSC washing buffer (0.2×SSC, 1% SDS), elute the RNA with RNase H digestion. The eluent RNAs were further reverse-crosslinked by irradiating with 254 nm UV in the presence of acridine orange for 15 minutes, and then used for subsequent library construction.