The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage

SummaryThe biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5\u2032 ends that vary in size. Methods to identify the 5\u2032 ends of transcripts will facilitate the discovery of new promoters and 5\u2032 ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano\u2013cap analysis of gene expression (nanoCAGE), a method that captures the 5\u2032 ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5\u2032 ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome