Investigating the role of RNA secondary structure and RNA-binding proteins in post-transcriptional gene regulation in Arabidopsis

M Reichel1, Z Zheng1, Y Liao1, G Wong1, M Rettel2, C Ragan1, M Evers1, M Hentze2, T Preiss1 and A Millar1

  1. Australian National University, Canberra, ACT 2601, Australia
  2. European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany

RNA-binding proteins (RBPs) and small RNAs (sRNAs), such as miRNAs, are key factors that orchestrate post-transcriptional gene regulation. For sRNAs, the degree of sequence complementarity it shares with potential target mRNAs is thought to be the decisive factor in target recognition and the strength of silencing, thus constituting the predominant basis of many sRNA target prediction programs. Here, we show that miRNA target genes with identical miRNA-binding sites are differentially regulated, meaning that strong complementarity is a pre-requisite, but not a guarantee of strong silencing. For a number of natural miRNA target genes, we have found sequence conservation that flanks the miRNA binding site which has the potential to form predicted stem-loop RNA secondary structures. Mutagenesis of these structures demonstrates that they are required for strong silencing, most likely by promoting miRNA-target recognition. Currently, we are trying to understand the mechanism by which these structures facilitate miRNA regulation and whether RNA-binding proteins (RBPs) are involved. Since relatively little is known about plant RBPs, we have taken a global approach termed mRNA interactome capture, to identify the RNA-binding proteome of Arabidopsis. We identified more than 700 proteins in total, 300 with high confidence, which we defined as the Arabidopsis mRNA interactome. Gene ontology (GO) analysis links approximately 75% of these proteins with RNA biology and includes the identification of proteins with canonical RNA-binding domains, validating many bioinformatically predicted RBPs. However, many proteins had no GO annotations associated with RNA biology, including major signalling proteins, cytoskeleton-associated proteins, membrane transporters and a host of other proteins implying the existence of many unknown RNA-protein interactions within a plant cell. This has provided novel insights into plant RNA biology and will act as a foundation resource for future studies investigating post-transcriptional gene regulatory mechanisms.