Funkce jaderných fosfoinozitidů a jejich vazebných partnerů v genové expresi

(ČESKÝ) Fosfoinozitidy jsou negativně nabité fosfolipidy s inositolovou hlavičkou, která může být fosforylovaná. Fosforylací inositolu vzniká sedm různě fosforylovaných forem fosfoinozitidů, které mohou být mono-, bis- nebo tris-fosforylované. Role cytoplasmatických fosfoinozitidů byly popsané v regulaci dynamiky buněčných membrán a cytoskeletu, v transportu membránových váčků, v funkci iontových kanálů a transportérů a produkci druhých poslů. Jaderné fosfoinozitidy se podílejí na posttranskripčních úpravách a exportu pre-mRNA, DNA transkripci a remodelování chromatinu. Zatímco cytoplasmatické funkce jsou dobře popsané, molekulární mechanizmy jaderných fosfoinozitidů v těchto jaderných procesech nebyly doposud dostatečně prozkoumány. V této práci jsme si kladli za cíl popsat lokalizaci fosfoinozitidů v jednotlivých funkčních kompartmentech jádra, což nám napomůže objasnit zapojení fosfoinozitidů do jaderných procesů. Dále jsme se zaměřili na identifikaci jaderných fosfoinozitidů zapojených do regulace genové exprese a objasnení detailního mechanismu interakce PI(4,5)P2 a PHF8 v regulaci transkripce ribozomálních genů. Dvěma nezávislými metodami jsme popsali lokalizaci fosfoinozitidů na jaderné membráně, v jaderných škvrnách, nukleoplasmě a jadérku. Tato rozšířená jaderná lokalizace naznačuje a i...(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

Function of nuclear phosphoinositides and their binding partners in gene expression

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's..

Nuclear actin filaments recruit cofilin and actin-related protein 3, and their formation is connected with a mitotic block

Although actin monomers polymerize into filaments in the cytoplasm, the form of actin in the nucleus remains elusive. We searched for the form and function of β-actin fused to nuclear localization signal and to enhanced yellow fluorescent protein (EN-actin). Our results reveal that EN-actin is either dispersed in the nucleoplasm (homogenous EN-actin) or forms bundled filaments in the nucleus (EN-actin filaments). Formation of such filaments was not connected with increased EN-actin levels. Among numerous actin-binding proteins tested, only cofilin is recruited to the EN-actin filaments. Overexpression of EN-actin causes increase in the nuclear levels of actin-related protein 3 (Arp3). Although Arp3, a member of actin nucleation complex Arp2/3, is responsible for EN-actin filament nucleation and bundling, the way cofilin affects nuclear EN-actin filaments dynamics is not clear. While cells with homogenous EN-actin maintained unaffected mitosis during which EN-actin re-localizes to the plasma membrane, generation of nuclear EN-actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-actin manifests in two mitotic-inborn defects—formation of binucleic cells and generation of micronuclei—suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis

Nuclear Phosphoinositides—Versatile Regulators of Genome Functions

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), modulate nuclear processes. We focus on PI(4,5)P2’s role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets—recently described nucleoplasmic PI(4,5)P2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide–protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies