Natural antisense transcripts (NATs) are non-protein coding RNAs that could play an important role in regulating the expression of their counterpart protein encoding sense transcript. Although NATs are widespread in most eukaryotic genomes, very little is known about their functions. This study focuses on gaining a better understanding of the function of NATs in Toxoplasma gondii, a pathogenic unicellular eukaryote. Previously, we 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, we identified the promoter of TgUlp1 NAT to be located within the 3’-region of its counterpart coding sequence. While TgUlp1 mRNA level was detected at a lower level throughout the life cycle of T. gondii, its NAT level was upregulated when the parasite converts from actively replicating tachyzoite form to slowly growing bradyzoite form. To investigate the effect of TgUlp1 NAT on the expression of its counterpart mRNA, we used a reporter system bearing TgUlp1 mRNA sequences and showed that the single-stranded TgUlp1 NAT and its in vitro RNase III processed products have the ability to lower the expression of the reporter system. Using a transgenic Dicer-knockout (TgDicer-KO) strain, we showed that TgDicer is required for the function of TgUlp1 NAT in vivo. The findings strongly suggest that the RNA interference pathway is necessary for the function of TgUlp1 NAT.

天然反义转录物（NAT）是非蛋白质编码的RNA，在调节其对应的编码有义转录物的蛋白质的表达中可能起重要作用。尽管NAT在大多数真核生物基因组中都很普遍，但对其功能知之甚少。这项研究的重点是更好地了解弓形虫（一种病原体单细胞真核生物）中NAT的功能。以前，我们表征了编码sumoylation中第一个定型酶的基因，称为泛素样蛋白酶1（Tg Ulp1），并表明TgUlp1的表达对于弓形虫的生命周期至关重要。有趣的是，TgUlp1的基因座也转录NAT物种。使用双重荧光素酶测定法，我们确定了TgUlp1 NAT的启动子位于其对应编码序列的3'-区域内。尽管在弓形虫的整个生命周期中都检测到较低水平的TgUlp1 mRNA水平，但是当该寄生虫从主动复制的速殖子形式转变为缓慢生长的缓殖子形式时，其NAT水平被上调。为了研究TgUlp1 NAT对其对应mRNA表达的影响，我们使用了带有TgUlp1 mRNA序列的报告系统，并显示了单链TgUlp1 NAT及其体外表达。RNase III加工的产品具有降低报告系统表达的能力。使用转基因切丁机（Tg Dicer-KO）菌株，我们表明Tg切丁机是体内TgUlp1 NAT的功能所必需的。这些发现强烈表明，RNA干扰途径对于TgUlp1 NAT的功能是必需的。