Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional ­inhibition. Mol Biol Cell

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel.
Molecular Biology of the Cell (Impact Factor: 4.47). 06/2005; 16(5):2395-413. DOI: 10.1091/mbc.E04-11-0992
Source: PubMed

ABSTRACT Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

Download full-text


Available from: James G Patton, Sep 27, 2015
48 Reads
  • Source
    • "AM-D treatment in higher eukaryotes leads to the condensation and subsequent separation of the FC and GC nucleolar regions [30]. Accordingly, in Dictyostelium different proteins leave the nucleolus in different ways after AM-D treatment which is consistent with the existence of nucleolar subcompartments [15] [16] [17]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during nucleolar disruption as a result of either AM-D treatment or mitosis support these subcompartments. A model for the AM-D-induced redistribution patterns is proposed.
    Biochemical and Biophysical Research Communications 12/2014; 433(4). DOI:10.1016/j.bbrc.2014.12.050 · 2.30 Impact Factor
  • Source
    • "Nucleophosmin is an important nucleolar protein supporting translation of ribosomal proteins and associated with the nucleolar fibrillar component. Its localization depends on the functional state of nucleoli28. Although the connection between nuclear and nucleolar distribution of ΔNp63 and nucleophosmin is unclear, localization of nucleophosmin can serve as an additional indicator of TP53-related effects in MCF10A cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.
    Scientific Reports 04/2014; 4:4663. DOI:10.1038/srep04663 · 5.58 Impact Factor
    • "Cells with reduced levels of USPL1 (identified by nucleolar coilin stain; Fig. 2A, arrowheads) still exhibited transcriptional activity at levels either similar to, or only slightly reduced, in comparison to control cells (coilin in Cajal bodies, arrows; Fig. 2A). This is consistent with the fact that we did not observe nucleolar cap formation upon knockdown of USPL1, which would be expected upon a general inhibition of transcription (Carmo-Fonseca et al., 1992; Shav-Tal et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cajal bodies are nuclear structures involved in snRNP and snoRNP biogenesis, telomere maintenance and histone mRNA processing. Recently, the SUMO isopeptidase USPL1 was identified as a Cajal body component essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its relation to Cajal bodies. We demonstrate that the levels of RNAPII-transcribed snRNAs are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalizes with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus our data indicate that USPL1 plays a key role in the process of RNAPII-mediated snRNA transcription.
    Journal of Cell Science 01/2014; 127(5). DOI:10.1242/jcs.141788 · 5.43 Impact Factor
Show more