Zheng Zihui, Reichel Marlene, Deveson Ira, Wong Gigi, Li Junyan, Millar Anthony A
Division of Plant Science, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia.
Division of Plant Science, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia
Plant Physiol. 2017 Jul;174(3):1764-1778. doi: 10.1104/pp.16.01898. Epub 2017 May 17.
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 genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; , , , , and were all poorly silenced, whereas and were strongly silenced. Curiously, this is consistent with previous genetic analysis defining and 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 and 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 /65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in 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 / as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159.
在植物中，微小RNA（miRNA）与靶标的互补性长期以来一直被认为是决定miRNA-靶标相互作用沉默结果的主要因素，尽管这种相互作用的效率在植物中很少得到评估。在这里，我们对七个拟南芥基因进行了体内沉默效率分析，所有这些基因都含有类似互补性的保守miR159结合位点。发现这些基因被miR159差异沉默； 、 、 、 、 和 均沉默效果不佳，而 和 则被强烈沉默。奇怪的是，这与之前将 和 定义为miR159主要功能靶标的遗传分析结果一致。miR159与靶标的互补自由能，以及由侧翼区域AU含量所确定的miRNA结合位点可及性，均无法完全解释miR159沉默效率的差异。相反，我们发现 和 均被预测在miR159结合位点附近含有独特的RNA二级结构。该结构由两个茎环（SL）组成，预计在裸子植物等进化距离较远的物种的 /65同源物中形成。功能分析发现， 中的RNA结构与强沉默效率相关；引入突变破坏任一SL会减弱miR159的作用效率，而引入互补突变以恢复SL，但不恢复序列，则可恢复miR159介导的强沉默。因此，似乎这种RNA二级结构将 /划定为miR159的敏感靶标，这支撑了拟南芥miR159狭窄的功能特异性。
