Formation Of Nuclear Structures Research Articles

Interaction, mobility, and phosphorylation of human orthologues of WD repeat-containing components of the yeast SSU processome t-UTP sub-complex

We previously proposed a dynamic scaffold model for inner nuclear structure formation. In this model, structures in inter-chromatin regions are maintained through dynamic interaction of protein complex modules, and WD repeat- and disordered region-rich proteins and others act as scaffolds for these protein complexes. In this study, three WD-repeat proteins, i.e., CIRH1A, UTP15, and WDR43, were found in the nuclear matrix fraction and speculated to be present in the human t-UTP sub-complex of SSU processomes. The results obtained as to their subnuclear localization, binding with each other, mobilities, and phosphorylation were: (i) the majority of these proteins fused with GFP are localized to the fibrillar center region in nucleoli. (ii) these 3 proteins bind directly with each other in vitro. (iii) the movement of these proteins is very slow in living cells and independent of rDNA transcription. (iv) His-CIRH1A is phosphorylated at Thr(131) by a mitotic Xenopus egg extract, and binding with GST-UTP15 and GST-WDR43 is suppressed. These findings and others suggest that these 3 WD proteins found in the matrix fraction bind directly with each other, bind tightly to fibrillar center regions, and comprise a part of the nucleolar structure. These results are also consistent with our dynamic scaffold model.

Diverse functions of nuclear non-coding RNAs in eukaryotic gene expression

Recent genome-wide analyses revealed that eukaryotic genomes are almost entirely transcribed, generating a large number of short or long non-protein coding RNAs (non-coding RNAs; ncRNAs). Rapidly accumulating experimental evidence suggests that ncRNAs are not just transcriptional noise, but have biological roles in gene expression. In this review, we focus on the functions of nuclear-localized ncRNAs including the spliceosomal small nuclear RNAs. These nuclear ncRNAs play diverse regulatory roles in a wide-range of nuclear reactions, such as transcription, precursor-mRNA (pre-mRNA) splicing, nuclear structure formation, nuclear trafficking, and chromatin remodeling. The regulatory functions of ncRNAs in these reactions are reinforced by target-site recognition through base-pairing or formation of an RNA/DNA triple helix. Recent studies revealed an unexpected linkage between the machineries for RNA interference (RNAi)-mediated gene silencing and pre-mRNA splicing. In addition, the biogenesis of some ncRNAs was found to overlap with the pathway of pre-mRNA splicing. Our understanding of the mechanisms of coordinated gene regulation in the nucleus has increased dramatically through studies on nuclear ncRNAs. A new paradigm of "ncRNA regulation" is now emerging.

Reduced Ran expression in Ran +/− fibroblasts increases cytokine-stimulated nuclear abundance of the AP-1 subunits c-Fos and c-Jun

Ran (Ras-related nuclear protein), a Ras family GTPase, is involved in multiple cellular functions, including the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing-exportation, and nuclear protein importation. Ran +/− embryonic stem (ES) cells were produced in an attempt to generate Ran null mutant mice. Even after an extremely large number of blastocyst injections, no Ran +/− chimeric mice could be generated. Ran +/− ES cell-derived fibroblasts showed reduced Ran protein expression, and manifested augmented nuclear abundance of AP-1 factors (c-Jun and c-Fos) upon cytokine stimulation. Our experiments demonstrated that intracellular Ran protein levels controlled the nuclear presence of certain transcription factors, such as c-Fos and c-Jun.

Ran Overexpression Leads to Diminished T Cell Responses and Selectively Modulates Nuclear Levels of c-Jun and c-Fos

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

Ran overexpression leads to a compromised transcription factor nuclear translocation and diminished T-cells response to TCR activation (35.8)

Abstract Ran (Ras-related nuclear protein) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 hours after their activation. Ran's function in the immune system was investigated. In transgenic T cells with actin promoter-driven Ran expression, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor (TCR) activation in vitro, compared with wild-type T cells. At the molecular level, transgenic T cells were defective in nuclear factor kappa-B (NFκB), activator protein 1 (AP-1) and nuclear factor of activated T cells (NFAT) nuclear translocation. Our experiments showed, for the first time, that Ran has important regulatory function in T-cell activation, and such function is probably related to its role in nuclear import of critical transcription factors.

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.