Document Type

Article

Publication Date

11-25-2025

Comments

This article is the author’s final published version in Journal of Biological Chemistry, Volume 301, Issue 12, 2025, Article number 110857.

The published version is available at https://doi.org/10.1016/j.jbc.2025.110857. Copyright © 2025 THE AUTHORS.

Abstract

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.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Language

English

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