A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes.

增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）是DNA复制工厂的保守组成成分，其与PCNA的相互作用可介导多种必需DNA复制酶向DNA合成位点的招募。目前学界尚未完整阐明此类复制工厂的结构与组成，对其开展更深入的研究将有助于我们理解基因组与表观遗传稳定性的维持机制。为全面鉴定与PCNA相互作用的蛋白集合，我们开发了一套基于人类细胞的双分子荧光互补（bimolecular fluorescence complementation, BiFC）筛选体系，用于筛选PCNA互作蛋白。该针对人类cDNA文库的类双杂交型互作蛋白筛选方法快速高效，其基于荧光的蛋白相互作用检测信号可实现互作克隆的便捷筛选；我们将该体系与下一代测序（next generation sequencing）技术相结合，以鉴定编码互作蛋白的cDNA序列。该方法可重复鉴定出已有报道的PCNA互作蛋白，更为关键的是，还成功鉴定出RNF7、Maf1及SetD3作为新的PCNA互作蛋白。我们通过从人类细胞提取物中进行免疫共沉淀（co-immunoprecipitation）实验，以及利用重组蛋白开展相互作用分析，验证了上述相互作用。上述结果表明，双分子荧光互补筛选体系是鉴定活哺乳动物细胞内蛋白质相互作用的有效工具。该方法具备高通量、易于自动化的优势，因此拥有广阔的应用潜力。鉴于其与PCNA的相互作用，我们推测Maf1、RNF7及SetD3可能参与DNA复制、DNA修复或相关生物学过程。