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 ₂ /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.

Graphical Abstract

HIGHLIGHTS

RIF1 AS is dynamically regulated by DNA damage and cell cycle signaling.

RIF1-L to RIF1-S isoform switch is associated with primary cancers.

SRSF1 acts directly on Exon 32 to promote RIF1-L expression.

S/K cassette expanded the RIF1 chromatin interactomes and stabilized phase separation.