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 ATGRP7 oscillates with a 24-h rhythm and transmits the rhythmicity generated by the central oscillator within the cell. ATGRP7 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 32P-labelled in vitro transcripts or synthetic RNA oligoribonucleotides comprising the binding site under study.