Working on this project:
Post-transcriptional gene regulation is essential to permit cells to adapt to changing environmental conditions. Regulation at the mRNA level is mainly performed bycis-regulatory elements encoded in the UTRs. These are recognized by trans-acting factors dependent on sequence and/or shape. Thus, structures within the UTRs modulate the localization, translation efficiency and stability of mRNAs by facilitating or preventing the binding of trans-acting factors. In accordance with this, the conservation of the structure of a UTR is an indicator of physiological function. Secondary structure prediction in combination with evolutionary conservation enables the identification of functional structured RNA motifs independent of the expression level of the respective mRNA. In the last funding period, we have used such an unbiased structure prediction to discover a tandem binding site for the RNA-binding protein Roquin in the 3’UTR of the UCP3 mRNA. Detailed mutational analyses allowed us to revise the consensus for Roquin binding sites. Using this new consensus, we discovered high-affinity binding sites in a large number of new target mRNAs. Many targets are cell type-specifically expressed or regulated and highlight additional functions of Roquin beyond its established role in the immune system. In the next funding period, we will discover new, conserved structured elements that modulate post-transcriptional gene regulation. Thus, we will establish a high-throughput screen for the parallel analysis of thousands of conserved 3’UTR structures in different cell types and under various stress conditions. The importance of trans-acting factors known to recognize mRNA structures will be tested globally for all functional candidates. The best candidates will then be characterized in detail. Furthermore, we will use the UCP3 tandem binding site as a model system to study Roquin binding kinetics.