A regulatory circuit based on AS-NMD of RNA-binding proteins mediates biological timing

The RRM protein AtGRP7 forms a molecular slave oscillator downstream of the circadian clock in Arabidopsis. AtGRP7 negative autoregulates by alternative splicing (AS) coupled to NMD. Further, AtGRP7 impacts a suite of transcripts in a phase-dependent manner, suggesting the AtGRP7 feedback loop transduces circadian timing information from the circadian clock to downstream targets. RNA-protein interaction is studied using single molecule techniques. We applied fluorescence correlation spectroscopy (FCS) to delineate the binding sequence of AtGRP7 using short dye-labelled oligonucleotides. Photoinduced electron transfer between the dye MR121 and guanosine was implemented to uncover conformational dynamics of synthetic RNA hairpins harbouring the AtGRP7 binding site upon contact with AtGRP7. This provides a molecular framework how binding of the proteins to their own pre-mRNAs could lead to a shift in splice site. We have identified a single RNP-1 arginine within the AtGRP7 RNA recognition motif that is crucial for in vitro binding and in vivo function. Notably, this very arginine is ADP-ribosylated by a novel type III effector protein of phytopathogenic Pseudomonas syringae. This suggests that disabling the RNA-binding activity of AtGRP7 may be part of the bacterial virulence mechanism.