A Role for Splicing Factors and the Transcriptional Machinery in the Pathophysiology of Fanconi Anemia,
Authors: Green Allison M , Wilson James B , Jones Nigel J , Kupfer Gary M. 
Published: October 2019

Abstract 3443

Introduction:

Fanconi anemia(FA) is an autosomal and X-linked recessive genetic disorder characterized by congenital defects, aplastic anemia, and a predisposition to cancer. At the cellular level, patients with FA display hypersensitivity to DNA crosslinking agents and increased levels of chromosomal instability. Because of these cellular phenotypes, the FA pathway has been thought to function in DNA damage repair response. Our data suggest that one of the ways in which the FA pathway maintains genomic stability within cells is by preventing aberrant transcription following DNA damage. This regulation may be accomplished through a connection to splicing factors or through direct interaction with the transcriptional machinery itself (RNAPII).

Methods:

siRNA transfections were used to reduce protein levels of splicing factors. Survival assays were performed to determine cell sensitivity to mitomycin C (MMC). Chromatin extractions were performed to determine chromatin loading of proteins. Immunoprecipitations were used to determine protein-protein interactions.

Results:

Previous studies in our lab have shown that siRNA mediated knock down of the splicing factors ASF/SF2 and SC35 but not SRp55 result in decreased levels of FANCD2 ubiquitylation following MMC treatment and hypersensitivity to MMC. We have since shown that depletion of ASF/SF2 and SC35 but not SRp55 also prevents proper FANCD2 chromatin loading following MMC treatment. As depletion of the splicing factor ASF/SF2 has also been demonstrated to increase cellular genomic instability through the formation of increased levels of R-loop structures, we next wanted to determine what effects increased levels of RNaseH would have on activation of the FA pathway in cells with decreased ASF/SF2 expression. Interestingly, RNaseH overexpression was able to partially rescue the decreased levels of FANCD2 ubiquitylation following MMC treatment and hypersensitivity to MMC seen in cells with decreased protein levels of ASF/SF2. As splicing has been shown to occur co-transcriptionally, we next went on to determine whether transcription occurs normally in cells lacking an intact FA pathway. We discovered that proper degradation of the hyperphosphorylated, transcription competent form of RNAPII in response to DNA damage is dependent upon an intact FA pathway as cells mutant in FANCA or FANCD2 show delayed RNAPII degradation following MMC treatment. Accordingly, we also saw a decrease in the levels of RNAPII interacting with FANCD2 in chromatin after similar, short term MMC treatments. This was accompanied by FANCD2 interaction with RNAPII and BARD1 in a FANCD2 ubiquitylation-dependent manner.

Conclusions:

These results suggest that the FA pathway may play a part in regulating transcription via a connection to splicing factors and through direct interaction with the transcriptional machinery itself as a means of initiating the DNA damage response.

Disclosures:

No relevant conflicts of interest to declare.