PIP2-Effector Protein MPRIP Regulates RNA Polymerase II Condensation and Transcription.

The specific post-translational modifications of the C-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (RNAPII) correlate with different stages of transcription. The phosphorylation of the Ser5 residues of this domain associates with the initiation condensates, which are formed through liquid-liquid phase separation (LLPS). The subsequent Tyr1 phosphorylation of the CTD peaks at the promoter-proximal region and is involved in the pause-release of RNAPII. By implementing super-resolution microscopy techniques, we previously reported that the nuclear Phosphatidylinositol 4,5-bisphosphate (PIP2) associates with the Ser5-phosphorylated-RNAPII complex and facilitates the RNAPII transcription. In this study, we identified Myosin Phosphatase Rho-Interacting Protein (MPRIP) as a novel regulator of the RNAPII transcription that recruits Tyr1-phosphorylated CTD (Tyr1P-CTD) to nuclear PIP2-containing structures. The depletion of MPRIP increases the number of the initiation condensates, indicating a defect in the transcription. We hypothesize that MPRIP regulates the condensation and transcription through affecting the association of the RNAPII complex with nuclear PIP2-rich structures. The identification of Tyr1P-CTD as an interactor of PIP2 and MPRIP further points to a regulatory role in RNAPII pause-release, where the susceptibility of the transcriptional complex to leave the initiation condensate depends on its association with nuclear PIP2-rich structures. Moreover, the N-terminal domain of MPRIP, which is responsible for the interaction with the Tyr1P-CTD, contains an F-actin binding region that offers an explanation of how nuclear F-actin formations can affect the RNAPII transcription and condensation. Overall, our findings shed light on the role of PIP2 in RNAPII transcription through identifying the F-actin binding protein MPRIP as a transcription regulator and a determinant of the condensation of RNAPII.

The F-Actin-Binding MPRIP Forms Phase-Separated Condensates and Associates with PI(4,5)P2 and Active RNA Polymerase II in the Cell Nucleus.

Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.

Limited Proteolysis-Coupled Mass Spectrometry Identifies Phosphatidylinositol 4,5-Bisphosphate Effectors in Human Nuclear Proteome.

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.

Nuclear phosphoinositides and phase separation: Important players in nuclear compartmentalization.

Nuclear phosphoinositides are recognized as regulators of many nuclear processes including chromatin remodeling, splicing, transcription, DNA repair and epigenetics. These processes are spatially organized in different nuclear compartments. Phase separation is involved in the formation of various nuclear compartments and molecular condensates separated from surrounding environment. The surface of such structures spatiotemporally coordinates formation of protein complexes. PI(4,5)P2 (PIP2) integration into phase-separated structures might provide an additional step in their spatial diversification by attracting certain proteins with affinity to PIP2. Our laboratory has recently identified novel membrane-free PIP2-containing structures, so called Nuclear Lipid Islets (NLIs). We provide an evidence that these structures are evolutionary conserved in different organisms. We hypothesize that NLIs serve as a scaffolding platform which facilitates the formation of transcription factories, thus participating in the formation of nuclear architecture competent for transcription. In this review we speculate on a possible role of NLIs in the integration of various processes linked to RNAPII transcription, chromatin remodeling, actin-myosin interaction, alternative splicing and lamin structures.