ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation [ZFP207_AID_TT-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 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). The coordination between the N-terminus and C-terminus of ZFP207 drives the formation of biomolecular condensate with U1 snRNP, which creates a molecular environment to promote spliceosome assembly by facilitating the interactions between U1 snRNP and other splicing regulators. Collectively, our study demonstrates the critical role of ZFP207-mediated phase separation in ensuring proper U1 snRNP function and spliceosome assembly. To elucidate the hypothesis of the depletion of ZFP207 impairs the efficiency of spliceosome assembly, we employed transient transcriptome sequencing (TT-seq) to assess splicing efficiency. This was achieved by determining the ratio of spliced reads to newly synthesized reads during a 10-min window of 4sU labeling. To minimize potential confounding effects from other RNA synthesis and processing processes, our analysis specifically focused on reads that spanned the boundary between the last nucleotide of the intron and the initial nucleotide of its adjacent downstream exon, and calculated the spliced-to-unspliced reads ratio to serve as a representative indicator of the splicing efficiency of the corresponding intron.