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.

• Klíčová slova
• MPRIP, PIP2, RNA polymerase II, phase separation, transcription,
• MeSH
• aktiny * metabolismus   MeSH
• fosfatasa lehkého řetězce myosinu genetika   metabolismus   MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• proteinfosfatasy genetika   MeSH
• RNA-polymerasa II * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny * MeSH
• fosfatasa lehkého řetězce myosinu MeSH
• MPRIP protein, human MeSH Prohlížeč
• proteinfosfatasy MeSH
• RNA-polymerasa II * MeSH

The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.

• Klíčová slova
• adenovirus, baculovirus, circovirus, herpesvirus, lamin, nuclear actin, nuclear cytoskeleton, papillomavirus, parvovirus, polyomavirus,
• MeSH
• aktiny metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• cytoskelet genetika   metabolismus   MeSH
• druhová specificita MeSH
• interakce hostitele a patogenu * MeSH
• jaderná lamina metabolismus   MeSH
• jaderný obal metabolismus   MeSH
• laminy metabolismus   MeSH
• lidé MeSH
• náchylnost k nemoci * MeSH
• regulace exprese virových genů MeSH
• replikace viru MeSH
• virové nemoci etiologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aktiny MeSH
• laminy MeSH

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.

• Klíčová slova
• MPRIP, PIP2, actin, nucleus, phase separation,
• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• aktiny metabolismus   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• glykoly farmakologie   MeSH
• lidé MeSH
• myosin typu I metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• RNA-polymerasa II metabolismus   MeSH
• subcelulární frakce metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• aktiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• glykoly MeSH
• hexamethylene glycol MeSH Prohlížeč
• MPRIP protein, human MeSH Prohlížeč
• MYO1C protein, human MeSH Prohlížeč
• myosin typu I MeSH
• RNA-polymerasa II MeSH
• zelené fluorescenční proteiny MeSH

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.

• Klíčová slova
• limited proteolysis, mass spectrometry, nucleus, phosphatidylinositol 4,5-bisphosphate, phosphoinositides,
• MeSH
• buněčné jádro metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• genová ontologie MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie * MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• proteolýza * MeSH
• proteom chemie   metabolismus   MeSH
• regulace genové exprese MeSH
• sekvence aminokyselin MeSH
• trypsin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositol-4,5-difosfát MeSH
• peptidy MeSH
• proteom MeSH
• trypsin MeSH

Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• exocytóza fyziologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• HeLa buňky MeSH
• kultivované buňky MeSH
• kůže cytologie   metabolismus   MeSH
• lidé MeSH
• mikrofilamenta metabolismus   MeSH
• myosin typu I metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• pohyb buněk MeSH
• tvar buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• myosin typu I MeSH

BACKGROUND: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. CONCLUSION/SIGNIFICANCE: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

• MeSH
• buněčné jádro metabolismus   MeSH
• DNA primery genetika   MeSH
• fenotyp * MeSH
• genotyp MeSH
• imunoprecipitace MeSH
• myosin typu I genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• plazmidy genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• Myo1c protein, mouse MeSH Prohlížeč
• myosin typu I MeSH
• protein - isoformy MeSH

Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

• Klíčová slova
• viruses, cytoskeleton, lamin, nuclear actin, nuclear lamina, nucleus,
• MeSH
• aktiny metabolismus   MeSH
• Baculoviridae metabolismus   patogenita   MeSH
• buněčné jádro metabolismus   virologie   MeSH
• cytoskelet MeSH
• Herpesviridae metabolismus   patogenita   MeSH
• herpetické infekce metabolismus   patologie   virologie   MeSH
• laminy metabolismus   MeSH
• lidé MeSH
• replikace viru * MeSH
• Retroviridae metabolismus   patogenita   MeSH
• retrovirové infekce metabolismus   patologie   virologie   MeSH
• sestavení viru * MeSH
• virové nemoci metabolismus   virologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aktiny MeSH
• laminy MeSH

BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

• MeSH
• adenosindifosfát metabolismus   MeSH
• aktiny metabolismus   MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• jaderné lokalizační signály MeSH
• kalmodulin metabolismus   MeSH
• karyoferiny metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myosin typu I chemie   metabolismus   MeSH
• myosiny chemie   metabolismus   MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosindifosfát MeSH
• aktiny MeSH
• jaderné lokalizační signály MeSH
• kalmodulin MeSH
• karyoferiny MeSH
• Myo1c protein, mouse MeSH Prohlížeč
• myosin typu I MeSH
• myosiny MeSH

Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.

• MeSH
• aktiny chemie   metabolismus   MeSH
• biologické modely MeSH
• buněčné jádro chemie   metabolismus   MeSH
• lidé MeSH
• mikrofilamentové proteiny chemie   metabolismus   MeSH
• oprava DNA MeSH
• replikace DNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aktiny MeSH
• mikrofilamentové proteiny MeSH

Nuclear myosin I (NMI) is a single-headed member of myosin superfamily localized in the cell nucleus which participates along with nuclear actin in transcription and chromatin remodeling. We demonstrate that NMI is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermiogenesis. Quantitative PCR and western blots demonstrate that the expression of NMI in tissues varies with the highest levels in the lungs. The expression of NMI is lower in serum-starved cells and it increases after serum stimulation. The lifespan of NMI is longer than 16 h as determined by cycloheximide translation block. A homologous protein is expressed in human, chicken, Xenopus, and zebrafish as shown by RACE analysis. The analysis of genomic sequences indicates that almost identical homologous NMI genes are expressed in mammals, and similar NMI genes in vertebrates.

• MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• exprese genu MeSH
• fylogeneze MeSH
• genetická transkripce MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• myosin typu I chemie   genetika   izolace a purifikace   metabolismus   MeSH
• myši MeSH
• obratlovci genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie nukleových kyselin MeSH
• sérum chemie   MeSH
• techniky amplifikace nukleových kyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• myosin typu I MeSH