Research Article
Marek Malecki, Bianca Malec
Abstract
In health and disease, biomolecules, which are involved in gene expression, recombination, or reprogramming have to traffic through the nucleoplasm, between Nuclear Pore Complexes (NPCs) and genomic DNA (gDNA). This trafficking is guided by the recently revealed Nuclear Routing Networks (NRNs).\r\nIn this study, we aimed to investigate, if the NRNs have established associations with the genomic DNA in situ and if the NRNs have capabilities to bind the DNA de novo. Moreover, we aimed to study further, if nucleoplasmic trafficking of the histones, rRNA, and transgenes’ vectors, between the NPCs and gDNA, is guided by the NRNs.\r\nWe used Xenopus laevis oocytes as the model system. We engineered the transgenes’ DNA vectors equipped with the SV40 LTA Nuclear Localization Signals (NLS) and/or HIV Rev Nuclear Export Signals (NES). We purified histones, 5S rRNA, and gDNA. We rendered all these molecules superparamagnetic and fluorescent for detection with Nuclear Magnetic Resonance (NMR), Total Reflection X-Ray Fluorescence (TXRF), Energy Dispersive X-Ray Spectroscopy (EDXS), and Electron Energy Loss Spectroscopy (EELS).\r\nThe NRNs span between the NPCs and genomic DNA. They form firm bonds with the gDNA in situ. After\r\ncomplete digestion of the nucleic acids with the RNases and DNases, the newly added DNA - modified with the dNTP analogs, bonds firmly to the NRNs. Moreover, the NRNs guide the trafficking of the DNA transgenes’ vectorsmodified with the SV40 LTA NLS, following their import into the nuclei through the NPCs. The pathway is identical to that of histones. The NRNs also guide the trafficking of the DNA transgenes’ vectors, modified with the HIV Rev NES, to the NPCs, followed by their export out of the nuclei. Ribosomal RNAs follow the same pathway.\r\nTo summarize, the NRNs are the structures connecting the NPCs and the gDNA. They guide the trafficking of the biomolecules between the NPCs and gDNA.\r\n