Nuclear functions of CFAP20 in transcription and replication [FLASH-seq]

Fine-tuning DNA replication and transcription co-occurrence is vital to avoid collisions between their machineries. This is especially relevant near promoters, where RNAPII initiates transcription and often arrests, forming R-loops. Arrested RNAPII poses a roadblock for DNA replication, which evolutionarily initiates near promoters. The mechanisms that salvage arrested RNAPII during elongation to avoid conflicts with incoming replisomes remain unaddressed. In this study, employing genome-wide and proteomic approaches, we identify and characterize CFAP20 as part of a protective pathway that rescues arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops specifically near promoters, displaying defects in replication origin firing and fork progression. Co-depletion of the Mediator coactivator complex or removal of RNAPII engaged with R-loops rescues these replication phenotypes. Therefore, we propose that CFAP20 salvages arrested RNAPII under conditions of high Mediator-driven transcription to promote RNAPII elongation, thereby preventing collisions with co-directional replisomes. To explore whether CFAP20 is an RNA-binding protein, we performed FLASH-seq in RPE1 cells being either CFAP20-KO (negative control) or CFAP20-KO expressing FLASH-BIO-CFAP20.