Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription.

RNA polymerase I (Pol I) transcription is essential for the cell cycle, growth and protein synthesis in eukaryotes. In the present study, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) is a part of the protein complex on the active ribosomal promoter during transcription. PIP2 makes a complex with Pol I and the Pol I transcription factor UBF in the nucleolus. PIP2 depletion reduces Pol I transcription, which can be rescued by the addition of exogenous PIP2. In addition, PIP2 also binds directly to the pre-rRNA processing factor fibrillarin (Fib), and co-localizes with nascent transcripts in the nucleolus. PIP2 binding to UBF and Fib modulates their binding to DNA and RNA, respectively. In conclusion, PIP2 interacts with a subset of Pol I transcription machinery, and promotes Pol I transcription.

UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity.

To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1ß-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis.

Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy.

Using quantitative evaluation of immuno-gold labeling and antigen content, we evaluated various automated freeze-substitution protocols used in preparation of biological samples for immunoelectron microscopy. Protein extraction from cryoimmobilized cells was identified as a critical point during the freeze-substitution. The loss of antigens (potentially available for subsequent immuno-gold labeling) was not significantly affected by freezing, while the cryosubstitution with an organic solvent caused a significant loss of antigens. While addition of water can improve visibility of some cell structures, it strengthened the negative effect of cryosubstitution on antigen loss by extraction. This was, however, significantly reversed in the presence of 0.5% glutaraldehyde in the substitution medium. Furthermore, we showed that the level of these changes was antigen-dependent. In conclusion, low concentrations of glutaraldehyde can be generally recommended for cryosubstitution rather than the use of pure solvent, but the exact conditions need to be elaborated individually for certain antigens.

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding.

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

Ultrastructural localization of actin and actin-binding proteins in the nucleus.

Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

Nuclear distribution of actin and myosin I depends on transcriptional activity of the cell.

As previous studies suggested, nuclear myosin I (NMI) and actin have important roles in DNA transcription. In this study, we characterized the dynamics of these two proteins during transcriptional activation in phytohemagglutinin (PHA) stimulated human lymphocytes. The stimulation led to strong up-regulation of NMI both on the mRNA and protein level, while actin was relatively stably expressed. The intranuclear distribution of actin and NMI was evaluated using immunogold labeling. In nucleoli of resting cells, actin was localized predominantly to fibrillar centers (FCs), while NMI was located mainly to the dense fibrillar component (DFC). Upon stimulation, FCs remained the main site of actin localization, however, an accumulation of both actin and NMI in the DFC and in the granular component was observed. In the nucleoplasm of resting lymphocytes, both actin and NMI were localized mostly in condensed chromatin. Following stimulation, the majority of both proteins shifted towards the decondensed chromatin. In transcriptionally active cells, both actin and NMI colocalized with nucleoplasmic transcription sites. These results demonstrate that actin and NMI are compartmentalized in the nuclei where they can dynamically translocate depending on transcriptional activity of the cells.

Nuclear actin and myosin I are required for RNA polymerase I transcription.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Immunolocalization of upstream binding factor and pocket protein p130 during final stages of bovine oocyte growth.

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (<1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2-3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (>3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

Regulation of ribosomal RNA synthesis during the final phases of porcine oocyte growth.

In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [3H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 microM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 microM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.