A Novel Regulatory Role for the Circadian Clock Protein TOC1 via RNA binding

The circadian clock enables plants to predict daily changes of external signals and synchronize them with internal processes, conferring enhanced fitness and growth vigor. The first described Arabidopsis circadian clock protein is TIMING OF CAB EXPRESSION 1 (TOC1), which functions in a transcriptional feedback loop with two myb transcription factors, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Previous studies have shown that TOC1 is a DNA-binding transcriptional repressor of CCA1 and LHY. However, the DNA motifs enriched amongst TOC1 targets share weak sequence similarity and lack consensus, suggesting that TOC1 regulates the expression of its targets through a novel mechanism. Here we show that the TOC1 protein binds directly to RNA via its conserved CCT domain. Using in vitro RNA selection, we identified an RNA motif that is recognized by the TOC1-CCT domain. The TOC1-CCT domain binds to this RNA sequence with nanomolar affinity determined by quantitative electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC). NMR experiments showed that two CCT fragments, CCT533-547 and CCT550-565, use basic residues to bind the RNA motif. Mutational analysis confirmed that lysyl and arginyl residues bind to RNA in a cooperative manner. Furthermore, transiently expressed wildtype and mutant TOC1 in protoplasts demonstrated that RNA binding activity of TOC1 is required for its function as a transcriptional repressor in vivo. Our results reveal a novel regulatory mechanism for TOC1 through RNA binding, suggesting that TOC1 might play key roles as a multi-function protein.