LS-2-O-3522 Phosphoinositide islets in the cell nucleus: a novel nuclear compartment?

Email of the presenting author: sobol@img.cas.cz The eukaryotic nucleus is recognized as a highly organized and well-orchestrated organelle, where nuclear architecture plays a role in gene regulatory networks, epigenetic regulation of gene expression, and RNA processing events. So far, mostly protein complexes have been considered as important in the three-dimensional nuclear organization. We introduce novel structures containing phosphatidylinositol 4,5-bisphosphate (PIP2) which seem to be of importance as well. It was previously described that PIP2 is present in interchromatin granule clusters (IGC). Recently, we published data on PIP2 involvement in nucleolar organization and transcription of genes coding rRNA. Based on this, we mapped PIP2-containing structures using pre-embedding immunolabeling and 3D electron tomography also in nucleoplasm. In addition to PIP2 detected in IGC and in the nucleolus (Figure 1a), PIP2-positive structures are present in nucleoplasm as 70-100 nm roundish “phosphoinositide islets” (Figure 1a, b). Using electron energy-loss microscopy and super-resolution light microscopy (SIM, STED), we demonstrated that they are composed mostly of phosphoinositides, and they are surrounded by chromatin. To explore the possible functions of the PIP2-containing islets, we mapped relative localization of PIP2 with a wide range of nuclear proteins involved in transcription, splicing, and chromatin organization using advanced immunogold electron microscopy and SIM. We demonstrate that at the periphery of the islets, PIP2 co-localizes or is located in close proximity to nascent transcripts as well as to the proteins engaged in Pol II transcription, mRNA splicing, and organization of chromatin. Direct binding, gradient fractionation, and mobility assays also showed nucleoplasmic interactions between PIP2 and proteins engaged in the chromatin remodelling and trancsription by Pol I and Pol II. Further, we revealed recruitment of some nuclear skeletal proteins into complexes with PIP2 using pull-down and 2D-electrophoresis assays. Taken together, our data allow us to suggest that newly observed PIP2-containing islets might play an important role in chromatin organization and nuclear architecture, thus participating in regulating gene transcription.