Mechanisms of Small RNA Degradation and Characterization of THO Complex Mutants in Arabidopsis
The expression of genes in eukaryotes is regulated at multiple layers. Transcriptional regulation and post-transcriptional regulation affect the mRNA levels of genes prior to protein translation. In my thesis work, I have worked on two research projects to study gene silencing at transcriptional and post-transcriptional levels in Arabidopsis. First, through a reverse genetic approach, I identified a nucleotidyl transferase HESO1 that functions in the degradation of microRNAs in the Arabidopsis hen1 mutant. As important regulators of gene expression, the accumulation of miRNAs affects the levels of their target mRNAs. I found that a mutation in HESO1 partially rescued the hen1 developmental defects, which result from decreased miRNA levels. The tailing of unmethylated miRNAs in hen1 by HESO1 promoted their degradation by an unknown nuclease, which differed from the one known to cause miRNA 3'-end truncation in hen1. I also confirmed the enzymatic activity of the HESO1 protein in vitro and found that 2'-O-methylation at the 3'-end of small RNAs completely blocked the activity of HESO1. These findings unveiled the protein implicated in small RNA tailing in hen1 and contributed to the knowledge of miRNAs degradation in Arabidopsis. Second, from forward genetic screens using two luciferase reporter lines that are regulated by DNA methylation, I isolated mutants with decreased luminescence and cloned three genes encoding subunits of the THO complex -- TEX1, HPR1 and THO5A. I found that DNA methylation was not affected in the mutants at either the LUCIFERASE transgene locus or most endogenous loci known to harbor DNA methylation. From mRNA sequencing, I identified genes that were up-regulated or down-regulated in tex1 or hpr1. I found that genes associated with dispersed repeats and inverted repeats were enriched among the down-regulated genes in tex1 and hpr1. Additionally, genes with H3K27me3 were enriched in both the up-regulated and the down-regulated genes in tex1 and hpr1. These findings provided insights into the potential targets of THO complex in Arabidopsis.