Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy.

Zihui Zheng,Gigi Wong,Junyan Li,Marlene Reichel,Anthony A Millar,Ira Deveson

doi:10.1104/pp.16.01898

Abstract

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159.

Highlights

In plants, microRNA-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, the efficacy of such interactions have rarely been appraised in plants
This defined the functional specificity of Arabidopsis miR159 being restricted to MYB33 and MYB65, and raised the question of the functional significance of miR159-mediated regulation of the additional bioinformatically predicted targets, including those that have a strongly conserved miR159 binding site (Allen et al, 2007, 2010)
Despite bioinformatics predicting miR159 to target approximately 20 genes in Arabidopsis, including eight MYB genes that contain conserved, highly complementary miR159 binding sites, miR159 appears functionally specific for MYB33 and MYB65 (Allen et al, 2007; Fig. 1A)

Summary

Introduction

MicroRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, the efficacy of such interactions have rarely been appraised in plants. This defined the functional specificity of Arabidopsis miR159 being restricted to MYB33 and MYB65, and raised the question of the functional significance of miR159-mediated regulation of the additional bioinformatically predicted targets, including those that have a strongly conserved miR159 binding site (Allen et al, 2007, 2010) This narrower functional specificity as defined by genetics has been found in other plant and animal miRNA systems, suggesting the functional scope of miRNA-mediated silencing is narrower than generally assumed (Seitz, 2009; Li et al, 2014a)

Methods

Results

Conclusion