Studies On The Function Of Promyelocytic Leukemia Protein Isoform Ⅱ

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are dynamic and heterogeneous nuclear protein complexes implicated in various important functions such as tumor suppression, viral defense and transcriptional regulation. Normal assembly of PML-NBs is of great importance in execution of their functions; however, the detailed molecular mechanisms have not been fully uncovered. PML is the structural component of PML nuclear bodies and has several nuclear splice isoforms which share a common N-terminal region but differ in their C-termini. These PML isoforms compose the trestle of PML nuclear bodies together. Previous studies have suggested that the coiled-coil motif within the N-terminal region is sufficient for PML nuclear body formation by mediating homo/multi-dimerization of PML molecules. To date, many relevant studies have been performed in PML-VI isoform, which lacks the C terninal region and is considered as a model representing the N-terminal region of PML. However the role of the C-termini of PML isoforms in this process has not been explored.In this study we foucs on the contribution of the C-terninal region of PML isoforms to PML-NB formation. We constructed the plasmids expressing the C-terminus of a varity of PML isoforms (GFP-PML-CT(15)), and then determined the subcellular localization of these fusion proteins by transfecting these plasmids to mammalian cells and microscopy detection. The results showed that the unique C-terminal domains of PML-Ⅱ and-V could target to PML-NBs independent of their N-terminal region, and formed nuclear bodies in the absence of endogenous PML. Further studies through immunoflurescence and FRAP experiments found that the C-terminal domain of PML-Ⅱ interacted transiently with the specific binding sites at PML nuclear bodies, while the C-terminal domain of PML-V exhibited a hyper-stable binding to PML bodies via homo-dimerization. This strong interaction was mediated by a putative a-helix in the C-terminus of PML-V. Moreover, the nuclear bodies formed through the C-terminal domain of PML-V had an ability to recruit multiple PML-NB-associated proteins, inculding Daxx and Sp100. These findings suggested that the C-termini of PML-Ⅱ and-V played important roles in the assembly, mobility and localization of PML-NBs.To further investigate the unknown binding partners of PML-CT2and reveal the new function of PML-Ⅱ isoform, we performed a yeast two-hybrid screening using PML-CT2as the bait and human leukocyte cDNA library as the prey. Among the positive clones, three candidate proteins (INTS4, UXT and ATXN3) were identified as the PML-CT2-interacting proteins. The specific interaction between PML-Ⅱ and these candidate proteins was further confirmed by Co-immunoprecipitation(Co-IP)-. Confocal microscopy detection and yeast-directed binding assay.Finally, the functional relationship between PML-Ⅱ and ATXN3was explored in the subsequent studies, we demonstrated that the overexpression of both WT and Mutant ATXN3alerted the intranuclear localization of PML-NB and the effect of ATXN3on PML-NBs relied on its de-ubiquitinating (DUB) activity, while PML-Ⅱ could antagonize this effect through the interaction between PML-CT2and ATXN3, which inhibited the DUB activity of ATXN3. These results indicated that the relationship of PML-II with ATXN3and revealed the specific function of PML-Ⅱ isoform.In summary, this study have shown that the C-termini of PML isoforms may play important roles in the assembly, mobility, localization and specific function of PML-NBs, which determines the diversity and o functional heterogeneity of PML nuclear bodies and provide new insights for understanding the regulation and assembly of PML-NBs.