Date of this Version
Meng Xie. The mechanism of small RNA biogenesis, degradation, and function in Arabidopsis. PhD dissertation, University of Nebraska-Lincoln. May 2014.
Eukaryotic small RNAs play important roles in many biological processes through sequence-specific RNA silencing. In plants, there are mainly two small RNAs triggering gene silencing: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The biogenesis and precise regulation of small RNA abundance are crucial for plant growth, development, genomic stability, and the resistance to both abiotic and biotic stresses. In this study, we used Arabidopsis thaliana, the model plant, to study the mechanism of RNA-directed DNA methylation (RdDM), in which siRNAs can trigger DNA methylation and gene silencing. In addition, we investigated the mechanism of miRNA biogenesis and degradation. For RdDM, we demonstrated that two SUPPRESSOR OF GENE SILENCING3 (SGS3)-like homologs named FACTOR of DNA METHYLATION 1 (FDM1) and 2 (FDM2) are required for de novo methylation established by RdDM. DNA methylation level and siRNA level are reduced significantly in fdm1 and 2 mutants. FDM1 and 2 are potential RNA-binding proteins with four domains: zinc-finger, XS, Coil-coiled, and XH domains. By studying the function of each domain, we propose that FDM1/2 can form a complex with other SGS3-like proteins and acts as a scaffold to stabilize the AGO4-siRNA-POL V transcripts, which is the essential structure to trigger de novo methylation. For miRNA biogenesis, we studied two protein factors: TOUGH and CDC5. We observed that TOUGH and CDC5 are required for proper function of DCL1 and miRNA processing. Moreover, TOUGH and CDC5 associate with key components in DCL1 processing complex, such as HYL1 and DCL1, which generate miRNAs. In addition, CDC5 interacts with DNA-dependent RNA polymerase II and is a positive transcription factor of genes encoding miRNAs. For miRNA degradation, we studied the mechanism underling miRNA uridylation catalyzed by one nucleotidyl transferase termed HESO1. Our data suggest that AGO1, the effector protein of miRNAs in Arabidopsis, associates with HESO1, which is necessary for the uridylation and degradation of unmethylated miRNAs by HESO1.
Advisor: Bin Yu