Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy

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.

MicroRNAs (miRNAs) are small 20- to 24-nucleotide (nt) RNAs that guide the RNA-Induced Silencing Complex to target mRNAs and mediate their silencing through a combination of transcript degradation and translational repression (Axtell, 2013). In plants, miRNAs have been shown to be involved in a multitude of critical developmental events and stress responses, and are often referred to as master regulators of gene expression. Central to understanding miRNA function has been identifying their target mRNAs (Sun et al., 2014). In plants, it is clear that high sequence complementarity between a miRNA and its target mRNA is compulsory for a miRNA-target interaction (Mallory et al., 2004; Schwab et al., 2005; Addo-Quaye et al., 2008; German et al., 2008), with most experimentally validated miRNA-target pairs having very few mismatches (Schwab et al., 2005; Liu et al., 2014). Consequently, miRNA-target complementarity has been the cornerstone of plant miRNA biology, determining miRNA target prediction (Dai and Zhao, 2011), the design of artificial miRNAs (amiRNAs; Schwab et al., 2005), the design of artificial miRNA decoys such as target MIMICs (Todesco et al., 2010), or the identification of endogenous target MIMICs (Karakülah et al., 2016). However, bioinformatic prediction of target genes often fails to accurately predict functionally relevant targets, where from numerous predicted targets, only a select few appear functionally significant (for review, see Li et al., 2014a). Likewise, it has been reported that amiRNAs with high complementarity to their intended targets perform with considerable variability in plants (Li et al., 2013; Deveson et al., 2013). Finally, different miRNA decoys that contain identical miRNA binding sites work with widely varying efficacies (Reichel et al., 2015). These and other observations argue that miRNA-target interaction is not simply a product of complementarity, but additional factors are required for functional miRNA-target interactions (Wang et al., 2015).

In animals, it has long been known that the contextual sequence features in which a miRNA-binding site resides can strongly impact silencing. For example, miRNA-binding site accessibility was shown to be important, where introduction of mutations to decrease predicted accessibility disrupted efficient regulation, with impacts being as strong as mutations within the binding site itself (Kertesz et al., 2007). Furthermore, it has been shown that for certain animal miRNA-target interactions, strong regulation only occurs when the binding sites are within specific sequence arrangements or contexts (Didiano and Hobert, 2006; Vella et al., 2004). Other factors impacting the efficiency of regulation include RNA-binding proteins (RBPs), which can either attenuate or facilitate the access of the miRNA to its binding site (Kedde et al., 2010). Factors such as these have been studied less in plants, but evidence is accumulating that sequence context of miRNA binding sites may also be important in plants. First, Gu et al. (2012) found a synonymous codon bias favoring AU-richness, and hence reduced RNA secondary structure, around predicted miRNA target sites in several plant species. Second, Li et al. (2012) have found that miRNA binding sites in Arabidopsis (Arabidopsis thaliana) are generally less structured than their flanking regions, indicating a preference for high accessibility. Indeed, Fei et al. (2015) found that target site accessibility may explain select regulation of only a few targets from a large number of predicted target genes. Therefore, it would be of interest to functionally test these potential factors on miRNA-target interactions.

In plants, the Arabidopsis miR159 family has been extensively studied as a model for plant miRNA-mediated gene regulation (Palatnik et al., 2003, 2007; Allen et al., 2007, 2010). The family has two major isoforms, miR159a and miR159b, which are strongly expressed throughout Arabidopsis (Palatnik et al., 2007; Li et al., 2016). Such expression is consistent with a loss-of-function mir159ab double mutant that displays strong pleiotropic developmental defects. In Arabidopsis, miR159 is bioinformatically predicted to regulate more than 20 targets, including eight genes encoding conserved R2R3 domain MYB transcription factors (Palatnik et al., 2007). Despite this, genetic analysis revealed that miR159-mediated regulation of only two of the predicted target genes, MYB33 and MYB65, account for the developmental defects of mir159ab, as all defects are suppressed in a myb33.myb65.mir159ab quadruple mutant (Allen et al., 2007). This defined the functional specificity of Arabidopsis miR159 being restricted to MYB33 and MYB65, but also 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). Curiously, this narrower functional specificity as defined by genetics has also 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).

For miR159, it is likely that multiple factors contribute to this apparent narrow functional specificity, including nonoverlapping transcriptional domains of the miR159 and MYB target genes (Allen et al., 2007), whether regulation of other targets is important under certain untested growth conditions or certain miR159 isoforms have become obsolete (Allen at al., 2010). However, one untested hypothesis is that MYB33 and MYB65 are more sensitive to miR159 regulation than the other MYB target genes. Recently, we have shown that factors beyond complementary govern the efficacy of the miR159-MYB33 silencing outcome (Li et al., 2014b). This not only included the miR159:MYB33 transcript stoichiometry, but also the sequence context of the miR159 binding site in MYB33. We showed that mutation of nts that immediately flank the miR159-binding site attenuated silencing to a similar extent to mutating nts within the binding site itself (Li et al., 2014b). This is further evidence that sequence complementarity alone does not guarantee strong miRNA regulation and that additional factor(s) are at play impacting miRNA-mediated regulation in plants.

Here, by carrying out in planta miR159 efficacy assays, we show that MYB33 and MYB65 are indeed much more sensitive to miR159 regulation than the other MYB genes with conserved miR159 binding sites. Neither flanking AU content nor predicted accessibility of the miR159 binding site underlie this sensitivity. Rather, MYB33 and MYB65 are shown to share a predicted RNA secondary structure consisting of two stem-loops that partially overlap with the miR159 binding site. Structure-function analysis demonstrates that both these stem-loops are required for efficient miR159 mediated silencing of MYB33. We hypothesize that having strong RNA stem-loops adjacent to a miRNA binding site may facilitate accessibility of the binding site to the miRNA, which in turn promotes efficient silencing of the target gene. Show more