拟南芥基因转录边界附近的小RNA新类群PASRs和TASRs的系统发掘及生物学功能研究

During recent years, two novel sRNA species, PASRs (promoter-associated small RNAs) and TASRs (termini-associated small RNAs), surrounding the transcriptional boundaries of genes were discovered in animals, fungi and bacteria. However, it remains unclear whether PASRs and TASRs exist in plants. If these small RNAs (sRNAs) indeed exist in plants, the biogenesis pathway(s) and the regulatory mechanisms of these sRNAs need to be uncovered experimentally. Recently, our bioinformatics analysis provided some hints to support the existence of PASRs and TASRs on the protein-coding genes in Arabidopsis (Arabidopsis thaliana). Based on these observations, we plan to perform a systematic search for these two novel sRNA species in Arabidopsis. By using the sRNA high-throughput sequencing data from certain mutants such as rdr and dcl, the biogenesis pathway(s) of PASRs and TASRs will be partially uncovered. Based on the sequencing data of DNA methylome and degradome, the regulatory mechanisms of PASRs and TASRs will be depicted. By utilizing T-DNA insertion (promoter regions) mutants and employing transgenic experiments, the biological functions of certain PASRs and TASRs will be deeply analyzed. The successful completion of this proposed project could serve as a basis for further functional studies on PASRs and TASRs. Our results will promote the understanding on the biogenesis pathways and the functions of the novel sRNA species in plants.

近年在动物、真菌和细菌中发现位于基因转录边界的两类小RNAs：PASRs（promoter-associated small RNAs）和TASRs（termini-associated small RNAs）。而目前对植物中是否真实存在PASRs、TASRs还缺乏系统分析和实验验证，更不清楚其产生、作用途径。申请人团队在前期分析中，发现拟南芥中这两类sRNAs的踪迹。基于该基础，我们将系统发掘拟南芥中的PASRs、TASRs；基于rdr/dcl等突变体的小RNA测序数据，揭示部分PASRs、TASRs的产生途径；基于甲基化、降解组等测序数据，研究部分PASRs、TASRs的作用机制；对特定基因启动子区T-DNA插入突变体及转基因等实验研究，深入揭示PASRs、TASRs的生物学功能。本项目的顺利实施，将为植物小RNA新类群的发掘和功能研究做出贡献。

近年在动物、真菌和细菌中发现位于基因转录边界的两类小RNAs：PASRs（promoter-associated small RNAs）和TASRs（terminus-associated small RNAs）。而目前对植物中是否真实存在PASRs、TASRs还缺乏系统分析和实验验证，更不清楚其产生、作用途径。本研究中，基于sRNA-seq数据，我们采用移动窗口算法，在拟南芥464个蛋白编码基因的启动子区域附近发现了PASR富集峰；在552个蛋白编码基因的转录终止位点附近发现了TASR富集峰。由此可见，以富集峰形式存在的PASRs和TASRs并非个例，这部分sRNA的存在可能具有一定的生物学意义。通过对分布在富集峰范围内的sRNA序列特征进行统计，我们发现：无论是PASRs还是TASRs，无论其分布在蛋白编码基因的正义链还是反义链，其序列长度大多分布在21—24 nt，并大部分以5’ A或5’ U起始。通过对sRNA-seq、dsRNA-seq和DNA甲基化测序数据的分析，我们提出了针对部分PASRs和TASRs的产生途径和作用模型，即依赖RDR2/6、DCL2/3/4的活性加工产生，其中一部分载入AGO4中，介导对host gene的位点特异性甲基化修饰。通过对特定PASR序列的超表达转基因实验，我们进行了相对深入的生物学功能分析。发现该条PASR超表达会引起种皮颜色加深、植株生长缓慢、抽苔时间延迟并显著降低了开花结实率。上述研究结果为植物转录边界附近非编码RNA的发现，以及生物学功能和分子作用机制研究打下了良好的基础。