Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Biochemistry & Molecular Biology, RNA-protein interaction, detection method, KISS, Dengue flavivirus, DDX6, PACT, mammalian, cell microarray screen, LONG NONCODING RNAS, SUBGENOMIC RNA, VIRUS, IDENTIFICATION, FLAVIVIRUSES, MECHANISMS, EXPRESSION, BIOLOGY, SYSTEM, BINDS, RNA−protein interaction, Animals, DEAD-box RNA Helicases, Flavivirus, Humans, Proto-Oncogene Proteins, RNA, Untranslated, RNA, Viral, Virus Replication, 03 Chemical Sciences, 06 Biological Sciences, 31 Biological sciences, 34 Chemical sciences

Abstract:

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.