Secondary structure of target mRNAs influences microRNA regulation in plants.

Secondary structure of target mRNAs influences microRNA regulation in plants.

Marlene Reichel, Zihui Zheng, Junyan Li and Tony Millar

Australian National University, Canberra, ACT, Australia 

In both plants and animals, microRNAs (miRNAs) have emerged as key regulators of gene expression. To date, the degree of sequence complementarity between a miRNA and its target mRNA is thought to be the decisive factor in target recognition and the degree of silencing. Consequently, complementarity constitutes the predominant basis of many miRNA target prediction programs. However, despite there being a multitude of computationally predicted targets, typically only a subset of these targets appear functionally relevant, implying that there are factors other than sequence complementarity influencing miRNA regulation. For instance, using the Arabidopsis miR159 system, we have shown that of the eight conserved MYB target genes who all have bioinformatically analogous miR159 binding sties, only MYB33 and MYB65 are strongly miR159-regulated, while the other targets are poorly regulated. Interestingly, MYB33/MYB65 contain highly conserved nucleotides flanking their miR159 binding sites, which are predicted to form highly similar, highly conserved RNA secondary structures containing two stem-loop regions (termed SL1 and SL2). Those structures are absent in other MYB targets that are poorly regulated by miR159. Disruption of the SL1 and SL2 structures in MYB33, without altering the miR159 binding site, resulted in strong perturbation of miR159 regulation. Conversely, compensatory mutations to restore the SL1 and SL2 structures, but not the wild-type sequence, re-established strong regulation of MYB33, demonstrating that these local RNA secondary structures confer strong miR159-regulation. This is the first time in plants that the secondary structure of target mRNAs has been shown to influence miRNA-mediated silencing, highlighting that factors beyond complementarity need to be taken into consideration. Currently, we are trying to understand the mechanism by which these structures facilitate miR159 regulation and whether RNA binding proteins are involved.