拟南芥中核苷酸转移酶活性及其在RdDM途径中的分子机理研究

Transcriptome studies in plants, animals, and fungi have revealed pervasive transcription in genomes and the presence of multitudes of noncoding transcripts, although biosynthesis, degradation and the functions of the noncoding transcripts are largely unknown. One major factor that stabilizes small RNA is methylation by the methyltransferase HUA ENHANCER1 (HEN1). Small RNAs uridylation is a widespread phenomenon in hen1 mutants and is often associated with their reduced accumulation, which implies that there are specific uridylation enzymes existing in cells. By reverse genetic analysis, we have identified 10 genes in Arabidopsis, whose products have putative nucleotide transferase activities. Our previous studies show that ntp6 mutants show increased 24 nt siRNA abundance, ntp6 ntp7 double mutants show increased DNA methylation level at RdDM loci. This project will study noncoding RNA uridylation activities of NTP6 ,NTP2,and NTP7, and determine if these proteins are involved in 24 nt siRNA biosyanthesis and the RdDM pathway. We will construct triple mutants of these three genes, and compare siRNA abundance and DNA methylation level by high throughput sequencing, and compare by qPCR the expression of RdDM loci between wild type and the triple mutants. We will also express these three genes in E. coli, purify the proteins and identify the uridylation activities of each. In addition, we will study the subcellular localization of these three proteins. The results of this project will help us to better understand the mechanism of noncoding RNA biosynthesis and turnover; and will also contribute to knowledge of epigenetic mechanism and genome stability.

真核生物的转录组含有大量非编码RNA，但其合成和降解机制知之甚少。HEN1是使小RNAs 3＇末端甲基化的酶，hen1突变体中小RNAs 3＇尿苷化,且丰度降低，说明细胞中存在使RNA尿苷化的核苷酸转移酶, 尿苷化引起RNA降解。本课题组前期在拟南芥中找到10个可能编码核酸转移酶的基因，其中ntp6突变体中24nt siRNA水平、ntp6ntp7双突变体中RdDM位点甲基化水平均较野生型中的高。本项目拟研究NTP6等基因尿苷化非编码RNA的功能，探索其在24nt siRNA的生物合成及RdDM途径中的作用。将构建3种基因的3突变体，比较野生型和3突变体中24nt siRNA及其前体的丰度，比较RdDM位点的表达和DNA甲基化情况；体外表达纯化3种基因的蛋白产物，检测其活性；研究其亚细胞定位。结果将丰富我们对非编码RNA合成及降解机制的理解，有助于对RdDM途径和基因组稳定性等知识的掌握。

拟南芥基因组中有10个可能的核苷酸转移酶（NTP）基因，这些NTP蛋白在各种类型的RNA代谢调控中的作用机制与功能仍待研究。RdDM是植物中一种重要的DNA甲基化方式，对基因表达起重要的调控作用。目前，NTP蛋白是否参与调控RdDM途径不得而知。本项目利用模式植物拟南芥为实验材料，系统研究了NTP蛋白的组织表达模式、亚细胞定位、体外酶活等生化特性。并利用T-DNA插入突变体和CRISPR/Cas9基因编辑技术，获得了包括ntp2、ntp6、ntp7在内的10个NTP单突变体以及ntp2 ntp6、ntp2 ntp7、ntp6 ntp7、ntp2 ntp6 ntp7 等高阶NTP突变体材料。并利用获得的NTP遗传材料，检测了NTP2、NTP6、NTP7对24 nt siRNA和DNA甲基化的影响。同时利用NTP与dcl2 dcl3 dcl4、dcl2 dcl3 dcl4 poliv的组合，初步研究了HESO1和NTP6对P4RNA的修饰和调控功能。此外，项目利用新开发的3’-RACE技术，重点揭示了NTP基因在pre-miRNA 加工修饰和降解的中的功能，完善了植物miRNA的加工代谢途径。在本项目的资助下，项目组系统探讨NTP2、 NTP6、 NTP7蛋白对24 nt siRNA和DNA甲基化的代谢调控作用；发现NTP蛋白参与依赖于Pol IV的非编码RNA（P4RNA） 的修饰和稳定性调控新机制；揭示NTP蛋白在pre-miRNA 加工修饰和降解的中的功能；发表高水平论文13篇（第一标注10篇），其中SCI收录文章6篇，JCR一区论文4篇；申请专利3项。