Koo Adenine Si-Hui, Jia Weiyan, Kim Sang Hwa, Scalf Mark, Boos Claire E, Chen Yuhong, Wang Demin, Voter Andrew F, Bajaj Aditya, Smith Lloyd M, Keck James L, Bakkenist Christopher J, Guo Lin, Tibbetts Randal S
Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, 1111 Highland Ave, Madison, WI 53705, USA.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave, Madison, WI 53706, USA.
bioRxiv. 2024 Nov 1:2024.10.29.619708. doi: 10.1101/2024.10.29.619708.
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/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.
RIF1（RAP1相互作用因子）在DNA双链断裂修复、DNA复制和细胞核组织中发挥多种作用。RIF1由外显子32（Ex32）的可变剪接表达为两种剪接变体，即RIF1长型（RIF1-L）和RIF1短型（RIF1-S），Ex32在RIF1 C端结构域（CTD）中编码一个富含26个氨基酸的丝氨酸/赖氨酸盒式肽。在这里，我们证明Ex32的包含受到DNA损伤和肿瘤发生的抑制，但在细胞周期的G/M期达到峰值。Ex32的剪接插入由包括SRSF1（其直接与Ex32结合）等正向调节因子以及PTBP1和SRSF3等负向调节因子催化。异构体蛋白质组学显示RIF1-L与MDC1的关联增强，RIF1-L增强了MDC1募集到电离辐射诱导的病灶。RIF1-L和RIF1-S还表现出独特的相分离和染色质结合特性，这些特性受CDK1依赖性CTD磷酸化的调节。这些综合发现表明，受调控的可变剪接影响RIF1在基因组保护和组织中的功能的多个方面。
