Paul Nakane Prize Lecture | FROM NUCLEOLAR MORPHOLOGY TO MOLECULAR REGULATION OF GENE EXPRESSION BY PHASE SEPARATION

P. Hozák | Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic

Processes such as gene expression or DNA repair are compartmentalized within eukaryotic nucleus, and nuclear environment contains dynamic membrane-less sub-compartments whose formation is prevalently driven by phase separation. It Apparently, 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. I will briefly recapitulate the history of my research and devote a majority of my talk to recent findings from our Prague laboratory. findings. We discovered the Nuclear Lipid Islets (NLIs) –globular ~100 nm structures containing PI (4,5) P2 (PIP2) at their periphery which associate with key transcription factors and showed that NLIs are crucial for efficient Polymerase II transcription. To decipher whether the NLIs surface recruits a transcription regulatory protein through PIP2 molecules in their surface, we employed a proteomic approach based on differential quantitative mass in combination with super-resolution microscopy. We identified more than 300 NLIs-associated proteins belonging to gene expression (53%) and pre-mRNA splicing (33%). Super resolution microscopy confirmed that some 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 features 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 by attracting proteins which are prone to form liquid-like particles.

This study was supported by the Czech Academy of Sciences (JSPS-20-06); Grant Agency of the Czech Republic (19-05608S, 18-19714S); IMG ASCR, v. v. i. (RVO: 68378050); by the COST Action CA19105 and MEYS CR (COST Inter-excellence LTC19048), and Czech-BioImaging projects (LM2018129 and LM2023050, funded by MEYS CR).