The scope and impact of post-transcriptional gene regulation in plants - microRNAs and the RNA-binding proteome of Arabidopsis

Abstract

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are key players in post-transcriptional gene regulation in eukaryotes, however challenges still exist in studying their role in plant biology. MiRNAs are a class of small RNAs that negatively regulate gene expression in plants and animals. Their function can be investigated by using miRNA decoys, which are non-coding transcripts with one or multiple miRNA binding sites that act by competing with the endogenous target mRNAs. In this thesis, I have developed miRNA SPONGES (SPs), which have been proven effective in animal systems, to inhibit miRNAs in Arabidopsis and compared them to MIMICs and short tandem target mimics, two commonly used decoy methods in plants. I have found that SPs are able to generate strong loss-of-function phenotypes, however the efficacy of all decoys varies dramatically depending on the targeted miRNA, demonstrating that no one approach can guarantee the strongest inhibitory outcome. Furthermore, I show that decoy methods, similar to RNAi approaches, can have unintended off-target effects, necessitating molecular analyses to ascertain their impact on closely related miRNAs. The reasons for the differences in decoy efficacy are still unknown, but likely involve poor recognition of the decoy by the miRNA. MiRNA-target recognition is still predominantly based on sequence complementarity; however, it is becoming increasingly clear that this factor alone is not a reliable indicator of the strength of miRNA-target interactions. Here, I have investigated factors beyond complementarity in the Arabidopsis miR159-MYB system. The miR159 family is predicted to target more than twenty genes, of which only MYB33 and MYB65 are strongly regulated. These two genes contain strong putative RNA secondary structures upstream the miR159 binding site, which appear absent in poorly regulated target genes. By mutating these structures in MYB33, I show that they are required for efficient miR159-mediated silencing. This demonstrates that target RNA secondary structure has a critical influence on miRNA regulation in plants and highlights the importance of factors beyond complementarity. Animal studies have shown that miRNA-target interactions can also be influenced by RNA-binding proteins (RBPs), however compared to miRNAs, RBPs are still poorly characterized, especially in plants. This is mostly due to their heterogeneity, which has rendered their study on a global scale challenging. In the last part of my thesis, I present the development of mRNA interactome capture for Arabidopsis, which allows the system-wide identification of proteins bound to mRNA in vivo. Using etiolated seedlings as source material, the approach identified more than 700 proteins in total, 300 of them with high confidence (False Discovery Rate below 1%). This has experimentally validated the RNA-binding activity of many bioinformatically predicted RBPs and identified a diverse set of novel plant RBPs. The latter group includes well-studied proteins such as signalling proteins, cytoskeleton-associated proteins and membrane transporters, as well as largely uncharacterized proteins such as ALBA and DUF1296 domain proteins, suggesting the existence of many unknown RNA-protein interactions. This study represents the first mRNA interactome in plants and provides a vast resource for future studies investigating the function of RBPs.