DNA-damage dependent isoform switching modulates RIF1 DNA repair complex assembly and phase separation

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The protein RAP1 interacting factor 1 (RIF1) plays crucial roles in DNA double-strand break repair, DNA replication, and nuclear organization.
• The precise mechanisms by which RIF1 executes its diverse functions remain incompletely understood.

Purpose of the Study:

• To investigate how alternative splicing of RIF1 contributes to its functional diversification.
• To elucidate the role of RIF1 isoforms in response to DNA damage and their implications in cancer.

Main Methods:

• Analysis of alternative splicing events in RIF1, focusing on a cassette exon (Ex32) in the C-terminal domain (CTD).
• Identification of splicing factors (SRSF1, SRSF3, SRSF7) regulating RIF1 alternative splicing.
• Isoform-specific proteomic analyses to identify binding partners and functional differences between RIF1-L and RIF1-S.
• Investigation of RIF1 CTD phase separation activity and chromatin retention dynamics.

Main Results:

• Alternative splicing of RIF1 Ex32 generates RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with distinct functional characteristics.
• DNA damage inhibits Ex32 splice-in, increasing RIF1-S expression, a phenomenon observed in primary cancers.
• RIF1-L preferentially associates with mediator of DNA damage checkpoint 1 (MDC1) and enhances MDC1 focus formation.
• The Ser/Lys-rich cassette in RIF1-L stabilizes CTD phase separation activity and enhances chromatin retention, modulated by CDK1 phosphorylation.

Conclusions:

• DNA damage-dependent alternative splicing of RIF1 is a key mechanism for functional diversification.
• RIF1 isoforms play distinct roles in genome protection, with RIF1-L contributing to DNA damage response and RIF1-S potentially linked to cancer progression.
• These findings provide novel insights into RIF1 regulation and its multifaceted roles in maintaining genome integrity.