Condensation of ZFP207 and U1 snRNP promotes spliceosome assembly [RIP-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. Firstly, we cultured ZFP207AID mESCs in medium containing 200 μM 4sU for 24 hours, then harvested the cells and crosslinked them with UV365 (200 mJ). We then performed ZFP207 bioRIP without RNase I digestion to ensure the integrity of U1 snRNA. Two sequential streptavidin beads pulldowns were performed to increase the signal-to-noise ratio. The eluted RNA was extracted, and reverse transcription was performed using a specific primer targeting the 3’ end of U1 snRNA. The full-length U1 snRNA was then amplified, and a library was constructed for deep sequencing.