| dc.description.abstract | Giardia lamblia, a simple yet intriguing protozoan parasite, challenges conventional paradigms in gene expression through its unique mechanism of trans-splicing for Heat Shock Protein 90 (Hsp90). As a primitive organism, Giardia exhibits a blend of prokaryotic and eukaryotic features, making its Hsp90 gene a captivating subject of study. Unlike other biological systems, where Hsp90 is conventionally expressed, Giardia stands as an exception, expressing Hsp90 through trans-splicing, as established by previous research (Nageshan, Roy et al. 2011).
This study delves into the molecular intricacies of Giardia’s trans-splicing process, aiming to unravel the mechanisms governing the expression of Hsp90. The investigation commences with the identification of potential protein factors associated with Hsp90 pre-mRNAs, HspN, and HspC. Utilizing RNA-protein pull-down assays coupled with mass spectrometry, a novel candidate, ATP-dependent RNA helicase (DHR1), emerges. This putative helicase interacts specifically with HspN pre-mRNA, suggesting a role in facilitating trans-splicing by unwinding secondary structures.
The exploration extends to the spatial organization of Hsp90 genes within the nucleus, addressing the intriguing question of how distantly located RNAs find each other for trans-splicing. Employing DNA-FISH technology, the study reveals co-localization of HspN and HspC loci, indicating a sub-compartment within the nucleus that may facilitate efficient trans-splicing. Surprisingly, other trans-spliced genes, such as Dynein genes, also exhibit spatial proximity, further supporting the existence of a specialized nuclear hub for chromatin-RNA interactions.
A unique aspect of HspN and HspC pre-mRNAs is their translatability, possessing complete open reading frames. The study explores the individual expression of HspN, supported by cytosolic localization observed through RNA-FISH. Immunoblot analysis and mass spectrometry provide evidence of HspN’s expression, with structural predictions suggesting potential functional roles, including an active ATPase activity.
Genetically modified Giardia cell lines serve as tools to validate the efficiency and accuracy of Hsp90 trans-splicing. Episomally expressed HspN/HspC undergoes trans-splicing with their endogenous counterparts, resulting in the production of hybrid-FlHsp90. Mutational analysis highlights the essential role of cis-elements in trans-splicing, where even minor mutations impede the process.
In conclusion, this comprehensive study unravels novel insights into the trans-splicing mechanism of Hsp90 in Giardia, identifying a potential RNA helicase, elucidating nuclear organization, revealing individual expression of HspN, and providing in vivo evidence of the efficiency and accuracy of trans-splicing. These findings not only contribute to our understanding of Giardia’s unique gene expression but also hold promise for the development of minimal splicing tools with broader applications. | en_US |
