In vivo Validation of Hsp90 Trans-splicing in Giardia lamblia: Highlighting the Role of Cis-elements

Giardia lamblia causes giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene of Giardia 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. (c) 2024 Elsevier Ltd. All rights reserved.