| Summary: | PTB (HnRNP I) is a multifunctional RNA binding protein which participates in a variety of RNA metabolic processes put together called as post transcriptional gene regulation. It interacts with shuttling hnRNPs L, K and E2 of the spliceosomal machinery and also with other RNA binding proteins like PSF, Raver1 and Raver2, which assists PTB in splicing. Based on the complexity of these processes and multifunctional nature of PTB, we hypothesized that; it might interact with various additional proteins not identified till date. Keeping this objective in mind, we set out to screen the custom made 18 day old mouse testes cDNA library in pGAD10 vector available in the laboratory, to hunt for novel interacting partners of PTB using the Clontech’s Matchmaker Gal4 yeast two hybrid system III. PTB1, the prototype of PTB was chosen and the above mentioned cDNA library was screened for novel PTB interacting partners. Twenty five large scale library transformations (spanning 8*106 independent clones) were performed and 99 putatives were obtained. By re-transformation of these library plasmids with bait construct to check for the interaction phenotype and eliminating bait independent activation of reporter genes and elimination of known false positives, only 5 clones were consistent with the interaction phenotype. All these library plasmids were sequenced with vector specific primers, ORF was identified and BLAST analysis for the identification of insert was done. Two of these clones encoded the partial CDS of mouse Protein Inhibitor of Activated STAT3-PIAS3. One of these encoded the partial CDS of mouse TOLL Interacting Protein-TOLLIP. The other two encoded the partial CDS of mouse importin-α and mouse hnRNP K, both of which were already known interacting partners of PTB. GST pull down assay and mammalian matchmaker co-immunoprecipitation was used for confirming the in vitro one to one physical interaction between PTB and these newly identified protein partners. Indirect Immunofloresence was used for demonstrating the co-localization of PTB and PIAS3 in Gc1Spg mouse spermatogonial cell line. The fact that PIAS3 an E3 SUMO ligase was picked up as an interacting partner of PTB was interesting and we hypothesized that PTB might be a sumoylation substrate. Towards this, we first resorted to the prediction of sumoylation consensus motif by using SUMOPLOT. PTB indeed was found to have sumoylation consensus sites. Subsequently, in vivo sumoylation of PTB was demonstrated, where in over expression of donor protein [SUMO-1] and acceptor protein [PTB] in RAG-1 mouse kidney cell line had resulted in the identification of an approximately 67 kDa slow moving SUMO modified myc tagged PTB band apart from the bulk of unmodified 57 kDa myc-PTB. This confirmed the fact that PTB is SUMO modified only at a single consensus target site in vivo and attempts are made to map this site of modification. SUMOylation regulates diverse biological processes in vivo ranging from nucleo- cytoplasmic shuttling, alteration of protein-protein interaction, DNA protein interaction etc. PTB shuttles rapidly between the nucleus and cytoplasm in a transcription sensitive manner and the translocation of PTB to the cytoplasm, happens under the conditions of cell stress, viral infections, apoptosis and exposure of cells to genotoxic agents like doxorubicin. Phosphorylation of PTB at Ser-16 residue has been shown to modulate the nucleo-cytoplasmic shuttling of PTB, albeit shuttling can also occur irrespective of this modification. Interaction of PTB with an E3 SUMO ligase-PIAS3 and the fact that it is SUMOylated in vivo, we hypothesize that K-47 residue present in the NLS/NES might be the most probable site of this SUMO modification and SUMOylation of PTB by PIAS3 might regulate the nucleo-cytoplasmic shuttling of PTB.
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