| dc.description.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. To examine the presence of Ts reaction products (trans-spliced mRNA and lariat by-product), we designed probes to specifically target nucleotides unique to the products and thus distinguish them from reaction substrates. Nanostring technology validated the presence of Ts junctional sequence as well as presence of lariat by-product after the in vitro trans-splicing reaction of the pre-mRNAs.
In addition, we have demonstrated that the 26 nucleotide complementary sequences, a unique feature in these pre-mRNAs, are necessary as positioning elements for in vitro self-splicing reaction by employing oligo inhibition assay as well as using mutant pre-mRNA substrates in in vitro trans-splicing reaction. Further, to understand the importance of the critical sequence elements for the in vitro Ts reaction, we performed site directed mutagenesis to delete 5’ splice site-GT, 3’ splice site-AG, branch point adenine and polypyrimidine tract alone and in different combinations and investigated the ability of the mutant pre-mRNAs to undergo self-splicing. Analysis of the Ts reaction products resolved in high resolution PAGE post incubation of the wild type and the mutant pre-mRNAs in different combinations in the Ts buffer (devoid of nuclear proteins) revealed several incorrectly sized products as compared to wild type pre-mRNAs. Sequencing of these products uncovered that in the absence of the critical nucleotides previously implicated in trans-splicing reaction, other cryptic nucleotides participate in trans-esterification reaction resulting in incorrectly spliced products. Therefore, our study highlights the importance of these critical nucleotide elements to ensure accurate Hsp90 self-splicing reaction.
We performed in silico secondary structure analysis of HspN and HspC introns. Our structure prediction results for HspC intron highlighted similarity with crystal structure of Oceanobacillus Group II intron. HspN intron structure prediction highlighted structural reduction as compared to Oceanobacillus group II intron owing to compact HspN intron. Our results suggest that Hsp90 trans-splicing of Giardia lamblia may be mechanistically similar to Group II splicing with the limited repertoire of spliceosomal protein components identified in Giardia only facilitating the fidelity of trans-splicing reaction.
Furthermore, we have also addressed the in vivo mechanistic aspects of Hsp90 gene trans-splicing. We propose that Hsp90 trans-splicing reaction in vivo may be dictated by physical proximity of the corresponding pre-mRNAs. We have shown that HspN and HspC ORFs physically interact with each other using high resolution Chromosome Conformation Capture (3C) technology which accounts for interactions between specific loci on a population scale.
In addition to Hsp90, two other genes Dhcβ and Dhcγ undergo trans-splicing based expression from different ORFs scattered across different chromosomes of G. lamblia. A distinctive feature in the pre-mRNAs arising from fragmented genes of Hsp90, Dhcβ and Dhcγ dispersed several hundred kbs apart on the same chromosome and some on different chromosomes is the presence of complementary sequences in the split introns of pre-mRNAs to seek and base pair with corresponding pre-mRNAs. We conjectured that there could possibly be a specialized sub-compartment in Giardia nucleus where all split genes could be co-localized by chromatin looping. Our 3C results showed that HspN also interacts physically with Dhcβ C-2 present 1700 kb apart on chromosome 5 and Dhcγ C-1 on chromosome 3. We also experimentally determined the presence of Scaffold/Matrix attachment regions (S/MARs) in between the split genes on chromosome 5 which may aid tethering of chromatin loop to Giardia nuclear matrix.
Overall, our study highlights the role of cis acting elements in the pre-mRNAs and nuclear organization to facilitate trans-splicing of heat shock protein 90 (Hsp90) in Giardia lamblia.
-Vinithra Iyer (Prof. Utpal Tatu’s lab) | en_US |
