Complex Formation between FANCD2 and the Splicing Factor SRSF1 Helps Prevent R-Loop Accumulation through mRNA Export Regulation

Introduction. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCI-FANCD2 (ID2) complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and R-loop regulation. R-loops are co-transcriptional RNA-DNA hybrids that form naturally during replication and transcription, but their persistent formation can drive genomic instability and have been postulated as an endogenous source of genotoxicity that contribute to the FA phenotype. However, the interplay between FA proteins and R-loops remains mechanistically ill-defined.

Disturbance of mRNA processing, export, and splicing has also been associated with R-loop-mediated genomic instability. The splicing factor SRSF1 (ASF/SF2) functions putatively in constitutive and alternative splicing. Besides its splicing function, SRSF1 participates in mRNA nuclear export through the Nuclear Export Factor 1 (NXF1) pathway. Disordered expression of SRSF1 reportedly plays a critical role in fostering the presence of R-loops. In addition, alterations in splicing factors have been associated with cancer and myelodysplastic syndrome (MDS), which clinically resembles FA. In the present study, we uncover a non-canonical role of FANCD2 and SRSF1 in the prevention of pathogenic R-loop formation via regulation of mRNA export.

Methods. Cells: Patient-derived FA-D2 mutant (PD20), corrected (D2), and ubiquitin-dead mutant (K561R) expressing cells, HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation and ubiquitination assays were performed using SRSF1 purified from bacteria and ID2 purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay, S9.6 immunofluorescence, and S9.6 slot-blot. Immunoprecipitation of mRNPs was used to detect ribonucleoproteins bound to mature mRNA. RNA FISH was used 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. Specifically, our results identify a physical and functional relationship between ID2 and SRSF1. First, using in vitro and cell-based techniques, we demonstrate that SRSF1 activates the FA pathway by binding ID2 and stimulating its monoubiquitination in an RNA-dependent manner. Furthermore, our data show that FANCD2 monoubiquitination is critical for NXF1-SRSF1 nuclear export complex formation and subsequent mRNA export. Importantly, cancer-associated SRSF1 mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, impaired mRNA export, accumulation of R-loops, and an FA-like cellular phenotype.

Conclusion. Collectively, our findings uncover a novel non-canonical mechanism of FANCD2 and SRSF1in the prevention of R-loop formation via facilitation of mRNA export, thus preventing genomic instability.

Clinical applicability. Our results reveal a novel mechanism for a non-canonical role of the FA pathway in prevention of genomic instability, shedding light upon the most basic cellular processes of the FA pathway, which is germane for delineating more generalized mechanisms of oncogenesis and genome maintenance. As R-loops accumulation is associated with FA, we propose that R-loops and R-loop processing proteins offer new avenues as relevant therapeutic targets not only for FA but also for other bone marrow failure syndromes marked by aberrant RNA metabolism and multiple cancers.

No relevant conflicts of interest to declare.