The evolutionary conservation of sequences within an mRNA indicates their functional importance. The high conservation of protein coding sequences is particularly evident. Sequence conservation within the untranslated regions of an mRNA is usually low; but when it occurs, it signals important sites of post-transcriptional regulation. mRNAs, like other RNA molecules, are able to form complex three-dimensional structures. Thus, the evolutionary conservation of an mRNA fold is likewise indicative of a functional site.
We identified new binding sites for Roquin proteins, using bioinformatic prediction of mRNA folding. Roquin proteins recognize mRNAs shape-specifically and sequence-independently. The binding of Roquin to such so-called CDEs (constitutive decay elements) accelerates mRNA decay (Fig. 1).
We have discovered a tandem CDE in the 3’UTR of the UCP3 mRNA (Fig. 2). Structural probing and mutational analysis confirmed the importance of the two stem-loops for mRNA destabilization. Roquin binding was confirmed in vitro and post-transcriptional regulation in vivo. In-depth mutational analysis of the tandem CDE allowed us to refine the binding preferences of Roquin and to discover a multitude of other CDEs in previously unknown target genes. In future research, we want to build a complete picture of Roquin binding preferences and the hierarchy of addressed target genes. Furthermore, we will investigate the interaction of Roquin with other RNA-binding proteins that compete for mRNA binding.