Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Dept. of Parasitology, Faculty of Science, BIOCEV, Charles University, Czech Republic.
J Mol Biol. 2024 Feb 15;436(4):168440. doi: 10.1016/j.jmb.2024.168440. Epub 2024 Jan 11.
Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.
蓝氏贾第鞭毛虫引起贾第虫病，是全球最常见的人类传染病之一。我们实验室之前的研究表明，蓝氏贾第鞭毛虫的 hsp90 基因分为两部分，即 hspN 和 hspC。这些分裂基因的独立前体 RNA 通过反式剪接结合，产生全长 Hsp90（FlHsp90）mRNA。参与基因的遗传操作对于理解这种反式剪接表达 Hsp90 的机制和意义是必要的。在这项研究中，我们在 G. lamblia 中进行了基于转染的 hspN 和/或 hspC 外源表达。我们电穿孔了一个含有 Avi 标记的 Hsp90 的 hspN 组件的质粒，并检查了它在 G. lamblia 中的命运。我们表明，外源性表达的 hspN RNA 与内源性表达的 hspC RNA 发生反式剪接，产生杂交 FlHsp90。我们通过突变分析强调了 cis-元件在这种反式剪接反应中的重要性。携带 hspN 内含子区域缺失的附加体质粒显示出对反式剪接反应的抑制。此外，外源性 hspC RNA 也遵循与外源性 hspN 相同的命运，而当与附加体质粒共转染时，它们彼此发生反式剪接。使用 eGFP 作为测试蛋白，我们已经表明 hsp90 基因的内含子序列可以指导相关外显子序列的反式剪接介导修复。我们的研究提供了 Hsp90 反式剪接的体内验证，显示 cis-元件的关键作用，并重要地突出了 hsp90 内含子序列作为最小剪接工具的潜力。
