PUB - Publikationen an der Universität Bielefeld

The clock-regulated small RNA-binding protein AtGRP7 (Arabidopsis thaliana glycinerich RNA-binding protein) influences its own circadian oscillations by negative autoregulation at the post-transcriptional level, presumably by binding to its own transcript. The present work analyses the RNA-binding mechanism of AtGRP7 and its closest homologue AtGRP8 at a molecular level. Definite binding sites within the AtGRP7 and AtGRP8 transcripts have been identified and binding of both proteins to these sequences has been demonstrated using synthetic oligoribonucleotides. Mutational analysis of the RNA and the proteins has uncovered nucleotides in the RNA targets and amino acids in the RNA recognition motif (RRM), respectively, that are crucial for binding affinity and specificity. Moreover, direct insights into the binding process have been obtained by the establishment of single molecule techniques such as fluorescence correlation spectroscopy and atomic force microscopy-based force spectroscopy. AtGRP7 requires extended singlestranded regions for target recognition and has influence on the RNA secondary structure. The AtGRP8 binding process exhibits two distinct steps since specific and unspecific binding events are detectable during target recognition. In addition to the known regulation of AtGRP8 by AtGRP7, a reciprocal regulation of AtGRP7 and AtGRP8 in vivo has been demonstrated in transgenic plants overexpressing AtGRP7 and AtGRP8, respectively (AtGRP-ox). Moreover, the AtGRP-ox plants show that AtGRP7 and AtGRP8 share a number of downstream target transcripts. These findings extend the current picture of the AtGRP7 slave oscillator by incorporating a second interdigitated feedback loop centred around AtGRP8. Furthermore, the relevance of the AtGRP7 RNA-binding activity for its in vivo function has been demonstrated. Mutation of a conserved RRM arginine (R49Q) leads to a severe reduction in binding affinity in vitro. Overexpression of AtGRP7, but not AtGRP7-R49Q in transgenic Arabidopsis plants leads to alternative splicing and concomitant decay of endogenous AtGRP7 transcript. This indicates that high affinity RNA binding is required for the negative auto-regulation of AtGRP7 in vivo. This is the first example for an RNP1 mutation showing a direct correlation of binding affinity in vitro and function in vivo. The alternatively spliced AtGRP transcripts have a premature termination codon and decay rapidly. To address the mechanism of this rapid degradation upf1 and upf3 mutant plants were analysed that are impaired in the nonsense-mediated decay (NMD) of RNAs. Indeed, the alternative AtGRP splice forms are increased in the upf mutant plants, indicating a previously unknown connection of the circadian clock and the NMD pathway.