背景:真核基因含有在mRNA成熟过程中通过剪接体机制去除的内含子。内含子给细胞带来巨大的能量负担;因此,它们必须在维持基因组稳定性和/或调节基因表达方面发挥重要作用。疟原虫寄生虫中的许多基因(>50%)含有预测的内含子,包括5'和3'非翻译区(UTR)中的内含子。然而,UTR内含子在疟疾寄生虫基因表达中的作用尚不清楚。
方法:在本研究中,开发了一种附加型双荧光素酶测定法,以评估由具有或不具有来自四个约氏疟原虫基因的5UTR内含子的启动子驱动的基因表达。为了研究5'UTR内含子对内源基因表达的影响,pytctp基因在编码区的N端被3xHA标记,使用CRISPR/Cas9系统产生具有或不具有5'UTR内含子的寄生虫。
结果:我们表明,与没有5'UTR内含子的启动子相比,具有5'UTR内含子的启动子在驱动基因表达方面具有更高的活性。结果在表达由具有或不具有5'UTR内含子的启动子驱动的HA标记基因(pytctp)的重组寄生虫中得到证实。基因表达的增强是内含子大小依赖性的,但不是DNA序列,例如内含子越长,表达水平越高。当来自一种约氏疟原虫菌株的启动子被引入不同的寄生虫菌株时,观察到类似的结果。最后,5个UTR内含子在不同的寄生虫发育阶段被交替剪接,表明寄生虫在各个发育阶段调节基因表达的积极机制。
结论:疟原虫5'UTR内含子以大小依赖性方式增强基因表达;在不同寄生虫发育阶段存在选择性剪接的mRNA,提示5'UTR内含子的选择性切片是调节寄生虫基因表达和分化的关键机制之一。
BACKGROUND: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5\' and 3\' untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown.
METHODS: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5\'UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5\'UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5\'UTR intron were generated using the CRISPR/Cas9 system.
RESULTS: We showed that promoters with 5\'UTR introns had higher activities in driving gene expression than those without 5\'UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5\'UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5\'UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages.
CONCLUSIONS: Plasmodium 5\'UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5\'UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation.