biostudies-literatureKoo ASNational Institutes of Health National Cancer InstituteNIA NIH HHSNIAID NIH HHSNHLBI NIH HHSAmerican Heart Association IncNational Heart Lung and Blood InstituteNational Institute of General Medical SciencesNational Institute on AgingNational Institute of Allergy and Infectious DiseasesUniversity of Wisconsin Carbone Cancer CenterYale School of MedicineNCI NIH HHSNIGMS NIH HHS110857https://www.ebi.ac.uk/biostudies/studies/S-EPMC12702018biostudies-literatureUnknown301(12)How RIF1 (RAP1 interacting factor 1) fulfills its diverse roles in DNA double-strand break repair, DNA replication, and nuclear organization remains elusive. Here, we show that alternative splicing of a cassette exon (Ex32) encoding a Ser/Lys-rich cassette in the RIF1 C-terminal domain (CTD) gives rise to RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with different functional characteristics. We demonstrate that RIF1-Ex32 splice-in is mediated by an exonic splicing enhancer that is recognized by the serine and arginine rich splicing factor 1 (SRSF1) and antagonized by SRSF3 and SRSF7. Exposure to DNA damage inhibited Ex32 splice-in, potentiated the association of SRSF3 and SRSF7 with RIF1 pre-mRNA, and caused an increase in RIF1-S protein expression, which was also observed across a diverse set of primary cancers. Isoform-specific proteomic analyses revealed RIF1-L preferentially associated with mediator of DNA damage checkpoint 1 (MDC1) and sustained MDC1 focus formation to a greater extent than RIF1-S. We further show that the Ser/Lys-rich cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by cyclin-dependent kinase 1-dependent phosphorylation of the RIF1 CTD in response to G₂ DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 alternative splicing contributes to RIF1 functional diversification in genome protection.The Journal of biological chemistryDNA-damage dependent isoform switching modulates RIF1 DNA repair complex assembly and phase separation.PMC12702018EJ5-GFPR01 AI07908725PRE1374149R35 GM126914R01 HL130724RF1 AG069483R01 CA180765P30 CA014520P30CA014520Scalf MBoos CEWang DKoo ASTibbetts RSBajaj AKeck JLGuo LKim SHVoter AFSmith LMJia WChen YBakkenist CJfalseDNA-damage dependent isoform switching modulates RIF1 DNA repair complex assembly and phase separation.How RIF1 (RAP1 interacting factor 1) fulfills its diverse roles in DNA double-strand break repair, DNA replication, and nuclear organization remains elusive. Here, we show that alternative splicing of a cassette exon (Ex32) encoding a Ser/Lys-rich cassette in the RIF1 C-terminal domain (CTD) gives rise to RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with different functional characteristics. We demonstrate that RIF1-Ex32 splice-in is mediated by an exonic splicing enhancer that is recognized by the serine and arginine rich splicing factor 1 (SRSF1) and antagonized by SRSF3 and SRSF7. Exposure to DNA damage inhibited Ex32 splice-in, potentiated the association of SRSF3 and SRSF7 with RIF1 pre-mRNA, and caused an increase in RIF1-S protein expression, which was also observed across a diverse set of primary cancers. Isoform-specific proteomic analyses revealed RIF1-L preferentially associated with mediator of DNA damage checkpoint 1 (MDC1) and sustained MDC1 focus formation to a greater extent than RIF1-S. We further show that the Ser/Lys-rich cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by cyclin-dependent kinase 1-dependent phosphorylation of the RIF1 CTD in response to G₂ DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 alternative splicing contributes to RIF1 functional diversification in genome protection.2025-01-01T00:00:00Z2025 Dec2026-06-30T03:25:19.749Z2026-06-30T03:20:40.35ZS-EPMC127020184148990110.1016/j.jbc.2025.110857