Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.
Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3′-end regions of transcripts (3′-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3′-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3′-end formation in the intestine.
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