Alternative splicing modulates chromatin interactome and phase separation of the RIF1 C-terminal domain.

RIF1 (RAP1 interacting factor) fulfills diverse roles in DNA double-strand break repair, DNA replication, and nuclear organization. RIF1 is expressed as two splice variants, RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S), from the alternative splicing (AS) of Exon 32 (Ex32) which encodes a 26 aa Ser/Lys-rich cassette peptide in the RIF1 C-terminal domain (CTD). Here we demonstrate that Ex32 inclusion was repressed by DNA damage and oncogenesis but peaked at G/M phase of the cell cycle. Ex32 splice-in was catalyzed by positive regulators including SRSF1, which bound to Ex32 directly, and negative regulators such as PTBP1 and SRSF3. Isoform proteomics revealed enhanced association of RIF1-L with MDC1, whose recruitment to IR-induced foci was strengthened by RIF1-L. RIF1-L and RIF1-S also exhibited unique phase separation and chromatin-binding characteristics that were regulated by CDK1-dependent CTD phosphorylation. These combined findings suggest that regulated AS affects multiple aspects of RIF1 function in genome protection and organization.