Functional Studies Of Arabidopsis Nucleotide Transferase NTPs In RdDM Pathway

A non-coding RNA(nc RNA) is a functional RNA molecule that is not translated into a p rotein. The main classes of regulatory non-coding RNAs include: si RNA, mi RNA, Pi RNA and long non-coding RNAs(lnc RNA), the stability of non-coding RNAs level plays important roles in epigenetics regulation.In eukaryotes, small RNAs uridine is a conservative RNA modification process, this process is associated with the degradation of small RNA. Small RNAs methylation enzyme HEN1(HUA ENHANCER1) is a methyltransferase which protects small RNAs from 3’-to-5’truncation and 3’uridylation by catalyzing 3’ terminal ribose 2’-O- methylation.This is a common phenomenon in small RNAs biosynthetic pathway. In the hen1 mutant, 3’end of small RNAs are tailed and truncated, thereby the level of small RNAs are decreased significantly, which suggests that there are nucleotidyltransferases existing in cells that can make small RNAs uridylation,and uridylation causes the degradation of small RNAs.To identify small RNAs uridylation enzymes, we use reverse genetics approaches to find 10 putative nucleic acids transferase gene in arabidopsis thaliana. Previous studies found that among these 10 genes, only the defects of HESO1 functions can recover the phenotypes of hen1 mutant partially. High-throughput sequencing found that under hen1 background, HESO1 mutation makes the mi RNA uridylation phenomenon reduced obviously and the amount of micro RNAs were increased in hen1heso1 plants comparing to hen1 plants. The results suggest that HESO1 is the enzyme that is responsible for micro RNAs uridylation in hen1 mutant, uridylation of micro RNAs leads to its degradation. But, despite heso1-1 is a null mutant, there are a certain level of uridylation phenomenon of micro RNAs in heso1-8hen1-1 double mutant, which suggests that there are other nucleic acid transferase enzyme in arabidopsis thaliana that has micro RNA uridylation activities.We then studied the micro RNA uridylation activities of the remaining nine genes, and found that the mutation of one gene, which is named as UTR1(URIDYLYLTRANSFERASE 1)(At2g45620), also showed reduces micro RNA uridylation level. The preference of URT1 and HESO1 for micro RNAs substrate is different, they have unique and cooperative activities in micro RNA turnover. The rest of the eight HESO1 and UTR1 homologous gene, we called NTP(NUCLEOTIDYL TRANSFERASE PROTEIN) genes. Previous studies of our lab have shown that the eight NTP gene have no significant role in micro RNAs uridylation. In order to find out if these genes play a role in the uridylation of other non-coding RNAs, we analysis the si RNA level of the eight NTP mutant, high-throughput sequencing found the amount of endogenous 24-nt si RNA level in ntp6 mutant increased significantly, suggesting that NTP6 genes may be involved in endogenous 24-nt si RNA biosynthesis in plant.24-nt si RNA is the key factors in RNA mediated DNA methylation pathway(Rd DM), Rd DM is an important way of methylation. Rd DM pathway contains two main stages: the generation of 24-nt si RNA and si RNA mediated DNA methylation and transcriptional gene silencing(TGS). In arabidopsis, there are three DNA-depended RNA polymerases(Pol II, Pol IV, Pol V) that are involved in 24 nt si RNA biosynthesis and si RNA directed TGS. Pol IV transcribes a long noncoding single-stranded RNA in Rd DM loci, single-stranded RNA is converted to double-stranded RNA by RDR2. DICER- LIKE 3(DCL3) processes double-stranded RNA into 24-nt si RNA. 24-nt si RNA was methylated by methyltransferase HEN1(HUA ENHANCER1), RISC(RNA-induced silencing complex) is then assembled. Polymerase Pol V produces a scoffold RNA, which recruits RISC to the Rd DM loci, DNA methyltransferase DRM2 was also recruited to Rd DM loci,leading to DNA methylation.According to our previous results, ntp6 mutants can lead to increased abundance of endogenous 24-nt si RNA in arabidopsis, which indicates that NTP6 may be involved in plant endogenous 24-nt si RNA biosynthesis pathway. Phyligenetic tree analysis shows that AT3g56320(NTP7), AT3g61690(NTP8) and AT2g40520(NTP2) have close phyligenetic relations with NTP6, this project will study the functions of NTP2, NTP6, NTP7, and NTP8, in order to find out their role in 24-nt si RNA biosynthesis pathway and Rd DM pathway.In this study, ntp2,ntp6,ntp7,ntp8 single mutants were obtained through T-DNA insertion, ntp2ntp6, ntp6ntp7, ntp7ntp8, ntp6ntp8 double mutants were obtained through genetic hybrid. We use real-time fluorescent quantitative PCR and Mcr BC-PCR methods respectively, to detect and compare the levels of gene expression and DNA methylation at Rd DM loci between ntps mutants and wild type Arabidopsis. Levels of genes expression at Rd DM loci were found to be decreased and DNA methylation levels were found to be increased in ntps mutant comparing to those in wild type Arabidopsis, suggesting that NTPs play a negative regulatory role in Rd DM pathway. It is possible that NTPs uridylate non-coding RNAs in 24-nt si RNA biosynthesis, which in turn affects the levels of endogenous 24-nt si RNA, and influences DNA methylation and gene expression at Rd DM loci. The results of this study show that NTP2, NTP6, NTP7 and NTP8 are involved in 24-nt si RNA biosynthesis pathway and play roles in Rd DM pathway.