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

Koo, A. S.-H.; Jia, W.; Kim, S. H.; Scalf, M.; Boos, C. E.; Chen, Y.; Wang, D.; Voter, A. F.; Bajaj, A.; Smith, L. M.; Keck, J. L.; Bakkenist, C. J.; Guo, L.; Tibbetts, R. S.

doi:10.1101/2024.10.29.619708

bioRxiv · 10.1101/2024.10.29.619708

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

Koo, A. S.-H.·Jia, W.·Kim, S. H.·Scalf, M.·Boos, C. E.·Chen, Y.·Wang, D.·Voter, A. F.·Bajaj, A.·Smith, L. M.·Keck, J. L.·Bakkenist, C. J.·Guo, L.·Tibbetts, R. S.

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Abstract

How RIF1 (RAP1 interacting factor) fulfills its diverse roles in DNA double-strand break (DSB) repair, DNA replication, and nuclear organization remains elusive. Here we show that alternative splicing (AS) of a cassette exon (Ex32) encoding a Ser/Lys-rich (S/K) 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 splicing factor SRSF1 and antagonized by splicing inhibitors 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 S/K cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by CDK1-dependent phosphorylation of the RIF1 CTD in response to G2 DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 AS contributes to RIF1 functional diversification in genome protection.

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Koo, A. S.-H., Jia, W., Kim, S. H., Scalf, M., Boos, C. E., Chen, Y., Wang, D., Voter, A. F., Bajaj, A., Smith, L. M., Keck, J. L., Bakkenist, C. J., Guo, L., Tibbetts, R. S.. 2024-11-01. Alternative splicing modulates chromatin interactome and phase separation of the RIF1 C-terminal domain. https://doi.org/10.1101/2024.10.29.619708

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