Investigating Natural Antisense Transcripts in Toxoplasma gondii

Natural antisense transcripts (NATs) are non-coding RNAs that can regulate the expression of their counterpart protein-coding transcript. While NATs are widespread in eukaryotic genomes, very little is known about their mechanism. Our study focuses on gaining a better understanding of the function of NATs in Toxoplasma gondii, a pathogenic unicellular eukaryote. We recently characterized the gene encoding the first committed enzyme in SUMOylation, named Ubiquitin-like protease 1 (TgUlp1), and showed that the expression of TgUlp1 is vital to the life cycle of T. gondii. Interestingly, the locus of TgUlp1 also transcribes a NAT species. Using a dual luciferase assay and RT-qPCR, we identified the promoters of TgUlp1 mRNA and NAT and measured their transcript levels in tachyzoites and bradyzoites. We found that TgUlp1 mRNA and NAT are differentially regulated at the transcriptional level via promoter activity and transcript turnover. Furthermore, the products of TgUlp1 NAT processed by RNase III retain the ability to lower the expression of reporters carrying TgUlp1 mRNA sequences, suggesting the involvement of RNA interference pathway. In Dicer-knockout (TgDicer-KO) and Argonaute-knockout (TgAgo-KO) transgenic strains, a higher level of TgUlp1 NAT, and much lower level of TgUlp1 mRNA was detected, suggesting that Dicer and Ago may be involved in maintaining TgUlp1 mRNA. Although we were unable to determine the direct effect of TgUlp1 NAT on mRNA, we showed that the introduction of TgUlp1 NAT by electroporation negatively affected the level of TgUlp1 mRNA. Consequently, regulation by TgUlp1 NAT would also affect TgUlp1 protein levels and ultimately the SUMOylation pathway in T. gondii which plays an important role in host cell invasion and cyst genesis. However, underlying mechanisms remain to be investigated