Mechanistic Insights Into Heat Shock Protein 90 Hsp90 Trans splicing in Giardia lamblia

Abstract

Hsp90 gene is conserved and encoded by a single ORF with none to many cis-spliced introns across the biological kingdom. Previous studies from our lab have shown that Hsp90 gene in Giardia lamblia has a split nature having two ORFs present 777 kB apart on chromosome 5. The two ORFs transcribe independently to generate individual pre-mRNAs which get stitched by a novel trans-splicing mechanism to generate the mature full length Hsp90 mRNA. In this study, we have reconstituted Hsp90 trans-splicing (Ts) reaction in vitro using purified pre-mRNA substrates with a view to understand the sequence elements and protein factors necessary for the trans-splicing reaction. We cloned partial sequences of HspN and HspC ORFs retaining the sequence elements such as 5 splice site, 3 splice site, branch point adenine, polypyrimidine tract and the 26 nucleotide complementary sequence elements which we have previously implicated in the trans-splicing of Hsp90 pre-mRNAs. Purified pre-mRNA substrates, in vitro transcribed from respective clones were investigated for their ability to undergo trans-splicing in vitro by employing three approaches namely Reverse-transcriptase polymerase chain reaction (RT-PCR), body labelled pre-mRNA based method and Northern blot to detect unique trans-spliced junction after incubation of pre-mRNAs in the Ts buffer in the presence and absence of nuclear extract. Interestingly, all three approaches confirmed the ability of pre-mRNAs to undergo self-splicing in vitro in a Mg2+ ion dependent manner. We have further validated the presence of self-spliced junction sequence using Nanostring technology, which is quantitative, free from any amplification bias and has higher sensitivity. Nanostring technology employs the use of sequence specific DNA reporter probes chemically linked to unique fluorescent barcodes and unique capture probes to specifically hybridize and detect the target...