The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.
RNA-protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA-protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein-protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA-protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA-protein interaction screening tool.
['Lemmens|Irma|I|0000-0002-1636-4584', 'Jansen|Sander|S|', 'de Rouck|Steffi|S|', 'de Smet|Anne-Sophie|AS|', 'Defever|Dieter|D|', 'Neyts|Johan|J|', 'Dallmeier|Kai|K|', 'Tavernier|Jan|J|']
D000818:Animals D053487:DEAD-box RNA Helicases D005416:Flavivirus D006801:Humans D011518:Proto-Oncogene Proteins D022661:RNA, Untranslated D012367:RNA, Viral D014779:Virus Replication
mammalian detection method KISS DDX6 PACT Dengue flavivirus RNA−protein interaction cell microarray screen