Transitive silencing of mobile small RNAs in Arabidopsis Pollen

To assay siRNA movement in the pollen grain, we took advantage of the known behavior of 22nt siRNAs to target transcripts for cleavage and initiate secondary siRNA biogenesis. Previous research has demonstrated that when targeted by a 22nt microRNA, a transcript containing a truncated non-fluorescent ~400 bp version of GFP (trGFP) will produce GFP secondary siRNAs that can target a second copy of a functional full-length GFP mRNA in trans. Therefore, we created a ~400 bp non-fluorescent version of trGFP with a 5' 22nt microRNA target site driven by the vegetative cell-specific KRP6 promoter and transformed this construct into a line homozygous for the sperm-specific functional full-length GFP driven by the MGH3 promoter. If cleaved by a 22nt microRNA, the vegetative cell-driven trGFP will be cleaved into secondary siRNAs, and if the RNA components of this system are mobile and transferred into the sperm cells, the sperm-specific fluorescence of GFP will be reduced. We used two versions of the microRNA target site, which included a mock target site that is not targeted by small RNAs, the target site of microRNA173 (which is a 22nt known to induce the production of secondary siRNAs). We demonstrate that when the 22nt microRNA173 target site is used in the vegetative cell transcript, a corresponding decrease in sperm cell fluorescence is observed. In the mock small RNA target site this reduction in fluorescence was not observed. To ensure the transitive silencing sperm cell transcripts is occurring via secondary small RNAs, we transferred this trGFP experiment into a dcl2/dcl4 mutant background that is defective in TE 21-22nt siRNA silencing. In dcl2/dcl4 double mutants with the trGFP system, we find that the GFP fluorescence of the mock target site transgene does not change, while the repression found in wild-type pollen with the 22nt microRNA173 target site is alleviated. The data for microRNA173 is particularly important, as DCL2 and DCL4 are not necessary for microRNA173 production, and therefore this demonstrates that specifically secondary siRNAs acting downstream of microRNA action are required to silence the sperm cell GFP. We further analyzed the production of sRNAs derived from GFP in pollen grains of transgenic lines expressing the 22nt miR173 or 22nt mock target sites inserted on the 5' region of the trGFP transgene in both wt and dcl2/dcl4 backgrounds. Overall design: Four small RNA sequencing libraries. There are two genotypes: wt Col and a dcl2/dcl4 double mutant. Each genotype has one of two truncated-GFP transgenes: one with a 22nt microRNA173 target site and one with 22nt ''mock'' target site not targeted by small RNAs.

为检测花粉粒中小干扰RNA（small interfering RNA, siRNA）的移动情况，我们利用了22nt siRNA靶向转录本并引发其裂解、进而启动次级小干扰RNA生物合成的已知特性。已有研究表明，当被22nt微RNA（microRNA, miRNA）靶向时，携带截短型非荧光绿色荧光蛋白（trGFP，约400bp）的转录本会生成GFP次级小干扰RNA，可反式靶向功能性全长GFP mRNA的第二拷贝。据此，我们构建了一段约400bp的非荧光trGFP序列，其5'端带有22nt miRNA靶位点，该序列由营养细胞特异性KRP6启动子驱动；随后将此重组载体转化至由精子特异性MGH3启动子驱动的功能性全长GFP纯合品系中。若该trGFP被22nt miRNA裂解，则会生成次级小干扰RNA；若该系统的RNA组分可移动并转移至精子细胞，则精子特异性表达的GFP荧光信号会减弱。我们设置了两种miRNA靶位点变体：一种是不被小RNA靶向的模拟（mock）靶位点，另一种是已知可诱导次级小干扰RNA生物合成的22nt miR173靶位点。实验结果显示，当营养细胞转录本携带22nt miR173靶位点时，可观察到精子细胞的GFP荧光信号显著降低；而在模拟靶位点组中，未观测到荧光信号减弱现象。为验证精子细胞转录本的传递型沉默由次级小RNA介导，我们将该trGFP实验体系引入了存在转座因子（Transposable Element, TE）相关21-22nt siRNA沉默缺陷的dcl2/dcl4双突变体背景中。在携带trGFP体系的dcl2/dcl4双突变体中，模拟靶位点转基因株系的GFP荧光信号无明显变化，而野生型（wild type, wt）花粉中由22nt miR173靶位点介导的荧光抑制现象被解除。miR173相关实验数据尤为关键：由于DCL2与DCL4并非miR173生物合成所必需的蛋白，因此该结果证明，仅需在miRNA作用下游发挥功能的次级小RNA，即可介导精子细胞GFP的沉默。我们还进一步分析了野生型（wt）和dcl2/dcl4背景下，分别携带插入于trGFP转基因序列5'端的22nt miR173靶位点或22nt模拟靶位点的转基因株系花粉粒中，源自GFP的小RNA生成情况。实验整体设计：共构建4个小RNA测序文库。实验包含两种基因型：野生型Columbia（Col）生态型以及dcl2/dcl4双突变体。每种基因型分别对应两种截短型GFP转基因变体：一种携带22nt miR173靶位点，另一种携带不被小RNA靶向的22nt模拟靶位点。