Abstract 6100: The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export
Authors: Olazabal-Herrero Anne , Liang Fengshan , Dutta Arijit , Huang Yuxin , Liang Zhuobin , Gupta Abhishek , Teng Yaqun , Lan Li , Chen Xiaoyong , Pei Huadong , Pillai Manoj , Sung Patrick , Kupfer Gary 
Published: April 2023

Background: Fanconi Anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer susceptibility. FA gene mutations are also widespread somatically in non-FA cancer patients. While the FA pathway participates in DNA repair, it is also linked to R-loop metabolism and regulation. R-loops are co-transcriptional RNA:DNA hybrids that form as a result of replication-transcription collisions that are enhanced due to DNA damage, but their aberrant formation drives genomic instability and is an endogenous source of genotoxicity. However, the interplay between FA proteins, DNA repair, and R-loops remains mechanistically ill-defined. Disturbance of mRNA processing, export, and splicing has also been linked to R-loop-mediated genomic instability. The splicing factor SRSF1 participates in mRNA export through the Nuclear Export Factor 1 (NXF1). Aberrant splicing factors expression has been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), which clinically resembles FA. In the present study, we study the coupling of the DNA damage response to the prevention of pathogenic R-loops via mRNA export regulation through the FA pathway and SRSF1.

Methods: Cells: FA-D2 mutant, corrected, and ubiquitin-dead mutant; HeLa. siRNA transfections to reduce SRSF1 and FANCD2 protein levels. Cell survival assays against DNA damaging agents. In vitro immunoprecipitation and ubiquitination assays using purified SRSF1 and ID2. DART assay, S9.6 immunofluorescence, and S9.6 slot-blot to measure R loops in cells. Immunoprecipitation of mRNPs used to detect mature mRNA-bound ribonucleoproteins. RNA FISH to measure mRNA export.

Results: In this study, we provide strong evidence that the FA pathway coordinates the prevention of R-loop formation and subsequent genomic instability through its interaction with splicing factors via regulation of mRNA export. First, we demonstrate that knockdown of SRSF1, which is known to result in increased R-loops, also is associated with diminished activation of the FA pathway. Second, the central FA complex ID2 binds to SRSF1 in cells and in recombinant protein assays. Further, we demonstrate that SRSF1 activates the FA pathway by binding ID2 and stimulates its monoubiquitination in an RNA-dependent fashion. This activity is associated with co-localization in cells at transcriptional-DNA damage inducible R loops. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, cancer-associated SRSF1 mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export and R-loops accumulation.

Conclusion: Our findings uncover a novel mechanism of FANCD2 and SRSF1 in the prevention of R-loop formation via coupling of mRNA export to the DNA damage response, thus preventing genomic instability.

Citation Format: Anne Olazabal-Herrero, Fengshan Liang, Arijit Dutta, Yuxin Huang, Zhuobin Liang, Abhishek Gupta, Yaqun Teng, Li Lan, Xiaoyong Chen, Huadong Pei, Manoj Pillai, Patrick Sung, Gary Kupfer. The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6100.