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: 4.55). 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.

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Available from: James G Patton, Aug 20, 2015
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    • "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]. "
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    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 · 2.28 Impact Factor
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    • "Instead, SFPQ–Flag (as a cotransfected marker) and endogenous PSPs relocated to perinucleolar caps, from which NEAT1_2 ncRNA was absent (Figure 1B). Paraspeckles reportedly display actinomycin D-induced disruption and the concomitant relocation of protein components (Fox et al, 2002; Shav-Tal et al, 2005). "
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    ABSTRACT: Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs.
    The EMBO Journal 09/2012; 31(20):4020-34. DOI:10.1038/emboj.2012.251 · 10.75 Impact Factor
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    • "When examining the functions of speckle proteins that redistribute to nucleolar caps upon Act D treatment, we found that most are implicated in alternative splicing, microRNA processing, or both. Proteins with these properties include EWS (product of the Ewing sarcoma breakpoint region 1 gene), heterogeneous nuclear ribonucleoproteins F, H, and K, p68 RNA helicase, TLS, and CBP20 (Saitoh et al., 2004; Shav-Tal et al., 2005; Iwamoto et al., 2008). Moreover, primary microRNAs have recently been observed to accumulate in speckles (Pawlicki and Steitz, 2009). "
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    ABSTRACT: The evolutionarily conserved Pat1 proteins are P-body components recently shown to play important roles in cytoplasmic gene expression control. Using human cell lines, we demonstrate that human Pat1b is a shuttling protein whose nuclear export is mediated via a consensus NES sequence and Crm1, as evidenced by leptomycin B (LMB) treatment. However, not all P-body components are nucleocytoplasmic proteins; rck/p54, Dcp1a, Edc3, Ge-1, and Xrn1 are insensitive to LMB and remain cytoplasmic in its presence. Nuclear Pat1b localizes to PML-associated foci and SC35-containing splicing speckles in a transcription-dependent manner, whereas in the absence of RNA synthesis, Pat1b redistributes to crescent-shaped nucleolar caps. Furthermore, inhibition of splicing by spliceostatin A leads to the reorganization of SC35 speckles, which is closely mirrored by Pat1b, indicating that it may also be involved in splicing processes. Of interest, Pat1b retention in these three nuclear compartments is mediated via distinct regions of the protein. Examination of the nuclear distribution of 4E-T(ransporter), an additional P-body nucleocytoplasmic protein, revealed that 4E-T colocalizes with Pat1b in PML-associated foci but not in nucleolar caps. Taken together, our findings strongly suggest that Pat1b participates in several RNA-related nuclear processes in addition to its multiple regulatory roles in the cytoplasm.
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