Nuclear Phosphoinositides and Phase separation: Nuclear Lipid Islets and their importance in regulation of RNA Polymerase II driven transcription.

0555841 - ÚMG 2022 RIV UA eng O - Others
Hozák, Pavel
Nuclear Phosphoinositides and Phase separation: Nuclear Lipid Islets and their importance in regulation of RNA Polymerase II driven transcription.
2021
R&D Projects: GA ČR GA19-05608S; GA ČR(CZ) GA18-19714S; GA MŠMT LTC19048; GA MŠMT(CZ) ED1.1.00/02.0109; GA MŠMT(CZ) EF16_013/0001775; GA MŠMT(CZ) LM2015062
Research Infrastructure: Czech-BioImaging - 90062
Institutional support: RVO:68378050
Keywords : Nuclear Lipid * RNA Polymerase II * transcription * PIP2 * Nucleus
OECD category: Cell biology

Processes such as gene expression or DNA repair are precisely compartmentalized within eukaryotic nucleus. Nuclear environment contains dynamic membrane-less sub-compartments whose formation is prevalently driven by Phase separation. During phase separation a multivalent weak interactions lead to aggregation of molecules into liquid-behaving droplets delimited from neighboring environment. The lack of membranous barrier ensures fast intake of components. Thus formation of phase boundaries provides the surface for spatiotemporal control contributing to the high rate kinetics of crucial processes such as transcription, ribosome maturation, splicing. Our laboratory discovered a nanoscale particles - Nuclear Lipid Islets (NLIs). NLIs are up to 100 nm nucleoplasmic foci containing PI(4,5)P2 (PIP2) at their periphery which associate with key Transcription factors. We showed that NLIs are crucial for efficient Polymerase II transcription. We further observed that nuclear PIP2 distribution is affected by 1,6 hexandiol treatment. The drug disturbs a weak hydrophobic interactions and is broadly used for dissolving phase separated structures. To decipher whether the NLIs surface recruits a transcription regulatory proteins through PIP2 molecules in their surface, we employed a proteomic approach based on differential quantitative mass spectrometry (qMS). In our experimental pipeline we analyzed the PIP2-containing nuclear fraction. Our approach combines qMS analysis with super-resolution microscopy visualization of candidate proteins providing valuable information about NLIs protein composition. We identified more than 300 NLIs-associated proteins belonging to gene expression (53%) and pre-mRNA splicing (33%). Super-resolution microscopy confirmed that candidate proteins form foci in nucleoplasm and associate with sub-population of NLIs. Further, our bioinformatical analysis of putative NLIs proteins revealed that majority of them contain Intrinsically Disordered Regions (IDRs). IDRs are known feature of proteins undergoing phase separation under in vivo and in vitro conditions. Moreover, we found that the vast majority of these proteins contain K/R rich motifs, which were previously shown as recognition sites for phosphoinositide (PIPs) binding. We hypothesize that NLIs may serve as a structural platform integrating RNA Polymerase II transcription and pre-mRNA splicing. In general, PIPs may spatiotemporally orchestrate processes in nuclear environment by attracting proteins which are prone to form liquid-like particles.
Permanent Link: http://hdl.handle.net/11104/0330299