RNA-protein Interaction Mediating Post-transcriptional Regulation in the Circadian System

Post-transcriptional control makes an important contribution to shaping transcript profiles of circadianly regulated genes. In
Arabidopsis thaliana
, the clock-regulated glycine-rich RNA-binding protein
AT
GRP7 oscillates with a 24-h rhythm and transmits the rhythmicity generated by the central oscillator within the cell.
AT
GRP7 negatively auto-regulates its own expression at the post-transcriptional level. In response to an elevated protein level, a shift to a cryptic 5′ splice site within the intron occurs, leading to an unproductively spliced transcript that rapidly vanishes due to its short half-life. This feedback regulation relies on direct binding of the RNA-binding protein to its own RNA. Here we describe the analysis of RNA-protein interaction in vitro employing recombinant RNA-binding protein and
32
P-labelled in vitro transcripts or synthetic RNA oligoribonucleotides comprising the binding site under study.