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 showed 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.

Automated summary

The study reveals the split nature of the Hsp90 gene in G. lamblia, with two ORFs separated on chromosome 5 and connected through a unique trans-splicing reaction. The presence of canonical nucleotide elements in independent pre-mRNAs suggests the involvement of spliceosomes in trans-splicing of Hsp90. Research also shows that an RNA helicase interacts with HspN pre-mRNA, and the trans-spliced genes of Giardia are spatially close in the nucleus.