The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Department of Genome Integrity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Applied Proteome Analyses Research Center PIGMOD Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Liběchov Czech Republic

Laboratory of Cancer Cell Architecture Institute of Biochemistry and Experimental Oncology 1st Faculty of Medicine Charles University Prague Czech Republic

Laboratory of Proteomics Institute of Biochemistry and Experimental Oncology 1st Faculty of Medicine Charles University Prague Czech Republic

Yale Cancer Biology Institute Yale University School of Medicine West Haven Connecticut United States of America

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Plasma membrane and nuclear phosphatidylinositol 4,5-bisphosphate signalling in cancer