A nucleolar role for the Fanconi anemia pathway protein, FANCI, in rDNA transcription

Abstract

Fanconi anemia (FA) is a disease of DNA repair because proteins mutated in patients with FA are impaired in removing interstrand DNA crosslinks. We propose a new nucleolar role for the FA protein, FANCI, in ribosome biogenesis based on extensive preliminary data. Ribosome biogenesis is the process of making ribosomes that initiates in the cell nucleolus with the transcription and processing of the pre‐ribosomal RNA (rRNA) followed by the assembly of mature ribosomes. Immunoaffinity purification of FANCI from HeLa cell nuclear extract followed by mass spectrometry revealed co‐purification with nucleolar proteins required for ribosome biogenesis, including PES1. Using subcellular fractionation, we have found that FANCI is indeed enriched in the nucleolus. In particular, the deubiquitinated form of FANCI is by far the most predominant form of nucleolar FANCI, even under conditions of DNA damage by mitomycin C, which stimulate monoubiquitination of FANCI. Immunoprecipitation revealed that deubiquitinated FANCI, but not its binding partner in DNA repair, FANCD2, is co‐immunoprecipitated by the nucleolar protein, PES1. To probe a possible nucleolar function for FANCI, we depleted FANCI in human cells and observed reduced transcription by RNA polymerase I (RNAPI). In addition, FANCI is co‐immunoprecipitated by the large subunit of RNAPI, indicating a physical and functional role for FANCI in efficient pre‐rRNA transcription. Finally, our results demonstrate novel regulatory mechanisms by deubiquitinases on the ubiquitination state of FANCI in the nucleolus. While our model does not contest the well‐established underlying DNA repair defect observed in cells from FA patients, it does suggest a dual pathophysiology for FA that includes defects both in DNA repair and in ribosome biogenesis.Support or Funding InformationThis work is funded by the NIGMS and NCI.