Specific nuclear sub-compartments that are regions of fundamental processes such as gene expression or DNA repair, contain phosphoinositides (PIPs). PIPs thus potentially represent signals for the localization of specific proteins into different nuclear functional domains. We performed limited proteolysis followed by label-free quantitative mass spectrometry and identified nuclear protein effectors of the most abundant PIP-phosphatidylinositol 4,5-bisphosphate (PIP2). We identified 515 proteins with PIP2-binding capacity of which 191 'exposed' proteins represent a direct PIP2 interactors and 324 'hidden' proteins, where PIP2 binding was increased upon trypsin treatment. Gene ontology analysis revealed that 'exposed' proteins are involved in the gene expression as regulators of Pol II, mRNA splicing, and cell cycle. They localize mainly to non-membrane bound organelles-nuclear speckles and nucleolus and are connected to the actin nucleoskeleton. 'Hidden' proteins are linked to the gene expression, RNA splicing and transport, cell cycle regulation, and response to heat or viral infection. These proteins localize to the nuclear envelope, nuclear pore complex, or chromatin. Bioinformatic analysis of peptides bound in both groups revealed that PIP2-binding motifs are in general hydrophilic. Our data provide an insight into the molecular mechanism of nuclear PIP2 protein interaction and advance the methodology applicable for further studies of PIPs or other protein ligands.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Division BIOCEV Laboratory of Epigenetics of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Průmyslová 595 252 50 Vestec Czech Republic

Laboratory of Applied Proteome Analyses Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Rumburská 89 277 21 Liběchov Czech Republic

Laboratory of Genome Integrity Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Microscopy Centre Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

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