质体非编码小RNA发生及调控作用机制

Small non-coding RNAs are short regulatory non-coding RNAs that contain important genome information. They were widely present in eukaryotes but not in prokaryotes. Chloroplast is considered to originate from symbiotic cyanophyta, and evolutionally positioned between eukaryotes and prokaryotes. However, the existence, biogenesis and function of the plastid-related small non-coding RNA are far from known. The applicant has been working on the biogenesis and function of small non-coding RNAs since 2001. And recently, he makes significant progress on chloroplast related small RNA. The applicant identified an interaction between an RNA binding proteins, RBP, and AGO, which facilitate the loading of small RNAs into AGO, and subsequently increase anti-viral resistance of plant. The mutation of rbp also decreases the fecundity of aphid. Further sub-cellular analysis demonstrated that RBP is located in the chloroplast. The AGO protein interacting with RBP also accumulates in chloroplast. With chloroplast extraction and small RNA library construction, the applicant identified three types of small RNAs that related to chloroplast: 29nt sRNAs that generated from chloroplast genome and localized in chloroplast, 21nt sRNAs that generated from nucleic genome but localized in chloroplast, and 20nt sRNAs that generated from chloroplast genome but localized out of chloroplast. Based on the function of RBP and small RNA libraries, the applicant proposes to study the biogenesis, modification of small non-coding RNAs related to chloroplast and examine their possible function and regulatory mechanism. The evolution and function of small non-coding RNAs will be explored by comparative study of RNA silencing system among prokaryotes, eukaryote-localized prokaryotes, and eukaryotes. Furthermore, this project will uncover the regulatory mechanism of small RNA in plastid and the bi-directionally regulation of gene expression between plastid and nucleic. Meanwhile, this study will also improve our understanding of small non-coding RNA in molecular level.

非编码小RNA是真核生物内广泛存在的重要遗传信息分子，原核生物不编码典型小RNA。植物叶绿体由原核蓝藻包吞进化而来，在进化上具有特殊地位，我们对质体相关小RNA研究甚少，造成对非编码RNA调控网络认知的漏洞。申请者发现RNA结合蛋白RBP可与AGO互作，帮助AGO结合sRNA增强植物抗性，同时该基因定位于叶绿体并在叶绿体正常生物过程中起重要作用，与之互作的AGO蛋白在叶绿体内也有富集。分离叶绿体发现三类特异sRNA：29nt叶绿体基因组产生叶绿体内作用，20nt叶绿体基因组产生叶绿体外作用和21nt核基因组产生叶绿体内作用。本项目将对这些新型非编码小RNA的生成、修饰及调控功能进行研究，比较叶绿体相关小RNA和植物典型小RNA及原核内类似小RNA的区别，探讨非编码小RNA的可能起源和进化。研究结果将发掘新的非编码RNA调控元件及抗性机制，为基因信息传递过程中非编码RNA调控研究提供新思路。

非编码RNA在真核生物内具有重要的作用和意义。叶绿体是重要的真核生物细胞器，我们对叶绿体细胞器相关的非编码小RNA还知之甚少，对于叶绿体sRNA介导的遗传信息传递方式和表达调控网络研究尚很缺乏。本项目分离提纯了野生型拟南芥叶绿体，发现了叶绿体基因组编码的sRNA与核编码sRNA在细胞质和叶绿体之间的穿梭分布；构建叶绿体编码sRNA的小串联模拟靶标载体，高效沉默细胞质中的sRNA，分析了叶绿体富集RNA参与衰老和高温胁迫过程的应答。通过免疫沉淀结合质谱分析技术，筛选获得与AGO2相互作用并在叶绿体定位的关键因子RNA结合蛋白（RNA binding protein，RBP），阐明了叶绿体关键蛋白RBP参与RNA沉默的发生机制。