TAS3-derived short-interfering RNAs confer cleavage of mRNAs in rice. TAS3-derived short-interfering RNAs confer cleavage of mRNAs in rice

Plant TRANS-ACTING SIRNA3 (TAS3)-derived short-interfering RNAs (siRNAs) include tasiR-ARFs, which are functionally conserved in targeting AUXIN RESPONSE FACTOR (ARF) genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sative). Small RNA-seq results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice degradome datasets, and some of these siRNA-target interactions were experimentally confirmed in rice tas3 mutants. Consistent with altered expression of target genes, phenotypic variations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA-target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether our study sheds new insights into TAS3 genes in plants and expand our knowledge about rice TAS3 siRNA-target interactions. Overall design: 12 samples in total, 3 replicates for WT, tas3a1, tas3b1,tas3c1

植物反式作用小干扰RNA3（trans-acting siRNA3, TAS3）衍生的短片段干扰RNA（short-interfering RNAs, siRNAs）包含反式作用siRNA-生长素响应因子（tasiR-ARF），这类小RNA在靶向生长素响应因子（AUXIN RESPONSE FACTOR, ARF）基因方面功能保守，同时还包含一类鲜有研究的非tasiR-ARF型siRNA。本研究对水稻（Oryza sativa）中的TAS3 siRNA进行了系统表征。小RNA测序（small RNA-seq）结果显示，绝大多数TAS3 siRNA属于非tasiR-ARF组，而tasiR-ARF仅占极小比例。对双子叶与单子叶植物中TAS3基因的系统发育分析表明，其siRNA生成区域在禾本科植物中高度保守，尤以稻亚科（Oryzoideae）为甚。通过分析水稻降解组（degradome）数据集，不仅鉴定出了tasiR-ARF的靶基因，还鉴定出了非tasiR-ARF型siRNA的靶基因；其中部分siRNA与靶基因的互作已在水稻tas3突变体中得到实验验证。与靶基因的表达变化相一致，相较于野生型水稻，三个TAS3位点的突变体均表现出表型变异。核糖体在TAS3 siRNA与靶基因互作中的调控作用进一步得到揭示：TAS3 siRNA介导的靶基因切割（尤其是tasiR-ARF靶向ARF2/3/14/15的过程）在水稻多聚核糖体（polysome）样本中广泛存在。综上，本研究为植物TAS3基因的研究提供了新视角，并拓展了我们对水稻TAS3 siRNA与靶基因互作的认知。整体实验设计：本研究共设置12个样本，野生型（WT）、tas3a1、tas3b1、tas3c1各组均设3次生物学重复。