Abstract
Small, about 20-30 nucleotides RNAs regulate various biological processes in most eukaryotes. One of the conserved roles of small RNAs is to identify invaded selfish genetic elements and induce transcriptional silencing through heterochromatin formation. The ciliated protozoan Tetrahymena use such mechanism to identify transposon-derived sequences in the germline micronuclear (MIC) genome and remove them from the somatic macronuclear (MAC) genome during the sexual reproduction. During this DNA elimination process ~12,000 internal eliminated sequences (IESs), many of which are related to transposons, are removed from the new MAC. It has been revealed that IESs are targeted for heterochromatin formation followed by DNA elimination by ~29-nt small RNAs, called scnRNAs. Although scnRNAs are produced from both IESs and MAC-destined sequences (MDSs) of the germline genome at the early sexual reproduction stages, target-directed small RNA degradation (TDSD) process degrades scnRNAs complementary to MDSs in the parental MAC prior to DNA elimination. While TDSD is the key process to specifically target IESs for DNA elimination, the molecular mechanism for TDSD remains poorly understood. The aim of my PhD project is to better understand that how the TDSD process is regulated in Tetrahymena. In my PhD study, I identified a novel gene, EMA2, which is exclusively expressed during sexual reproduction and required for completing DNA elimination. Northern blot and small RNA sequencing analysis revealed that TDSD is severely affected in the absence of EMA2. Ema2 protein possesses a SP-RING domain, which is shared among some SUMO E3 ligases, directly interacts with the SUMO E2 enzyme Ubc9 and is required for the accumulation of SUMOylated proteins during sexual reproduction, all indicating that Ema2 is a SUMO E3 ligase. I further found that Ema2 is necessary for the SUMOylation of the conserved transcriptional regulator Spt6 and for the genome-wide non-coding RNA transcription from the parental MAC that we previously proposed to be a prerequisite for TDSD. Altogether, I propose that Ema2p-dependent SUMOylation of Spt6p is necessary for the non-coding RNA transcription from the somatic genome and thus for TDSD.