The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia

Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5’SS-GU, 3’SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we show that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.
Author summary Giardia lamblia causes the most common enteric disease called Giardiasis worldwide in humans and animals. Giardia is an intriguing model organism to study molecular evolutionary processes owing to its peculiar position, at the transition of prokaryotes and eukaryotes. Previous studies from our lab have shown a unique mode of expression of the Hsp90 gene, which is fragmented into two halves and remotely spaced in the Giardia genome. We showed that pre-mRNAs arising from the distant genes undergo molecular stitching by trans-splicing to generate the mature message. This process of molecular stitching of Hsp90 at the RNA level is unique to Giardia and is ill explored. The current study sheds light on the mechanisms of this molecular jugglery unique to this neglected parasite. In this study, we describe the role of nuclear architecture in bringing the fragmented genes, which are otherwise far apart, in close proximity to facilitate their molecular stitching. In addition, we highlight the role of protein factors in orchestrating this molecular feat. Our results point to the unique mechanism(s) which can serve as potential targets to develop specific treatments against this important pathogen.