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

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• RIF1 (RAP1 interacting factor) is crucial for DNA repair, replication, and nuclear organization.
• RIF1 exists as two splice variants, RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S), due to alternative splicing of Exon 32 (Ex32).
• Ex32 encodes a peptide in the RIF1 C-terminal domain, potentially modulating protein function.

Purpose of the Study:

• To investigate the regulation of Ex32 alternative splicing in RIF1.
• To characterize the functional differences between RIF1-L and RIF1-S isoforms.
• To understand how alternative splicing impacts RIF1's role in genome protection.

Main Methods:

• Analysis of Ex32 inclusion levels under various conditions (DNA damage, cell cycle).
• Identification of splicing factors regulating Ex32 splicing (e.g., SRSF1, PTBP1, SRSF3).
• Isoform-specific proteomics and biochemical assays to compare RIF1-L and RIF1-S.

Main Results:

• Ex32 inclusion is repressed by DNA damage and oncogenesis, peaking during G2/M phase.
• Splicing factors SRSF1, PTBP1, and SRSF3 directly regulate Ex32 splice-in.
• RIF1-L shows enhanced association with MDC1 and strengthens its recruitment to DNA damage sites.
• RIF1 isoforms display distinct phase separation and chromatin-binding properties modulated by CDK1 phosphorylation.

Conclusions:

• Regulated alternative splicing of RIF1 Exon 32 fine-tunes RIF1 function in response to cellular cues.
• RIF1-L and RIF1-S isoforms possess distinct properties influencing DNA repair and genome organization.
• Alternative splicing is a key mechanism controlling RIF1's multifaceted roles in maintaining genome integrity.