Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

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. INTRODUCTIONThe nucleus is a dynamic organelle consisting of interacting chromosomal and protein compartments. One of the major pathways of nuclear translocation is the movement of preribosomal particles from the nucleolus into the cytoplasm for the assembly of functional ribosomes. The main nucleolar functions involve RNA polymerase (pol) I transcription, posttranscriptional maturation of pre-rRNA transcripts and their subsequent assembly with ribosomal proteins into preribosomal particles. Other functions have been attributed to the nucleolus (for reviews, see Carmo-Fonseca et al., 2000;Olson, 2004b) and include the processing of RNA pol III transcripts, RNA editing, sequestration of cell cycle components in yeast, and Mdm2 protein in mammalian cells. The localization of telomere proteins and telomerase RNA in nucleoli suggests a role for the nucleolus in aging.Nucleolar components are found in all cells and tissues although the size, shape, and number of nucleoli may change depending on the species, cell type, and functional state. Transmission electron microscopy (TEM) has revealed three major structures within nucleoli: fibrillar centers (FC), dense fibrillar components (DFC), and the granular component (GC; for reviews, see Busch and Smetana, 1970;Goessens, 1984;Shaw and Jordan, 1995;Scheer and Hock, 1999). rDNA transcription units are found in the FC and consist of tandem repeats of these genes. rRNAs are harbored within the DFC and are processed there. It is therefore thought that rRNA transcription occurs at the interface betw...

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

“…These experiments showed that only CF I m 68 and PSP1 were present in the same perinucleolar cap structures upon inhibition of transcription. This result is consistent with the observation that all other paraspeckle proteins known so far (PSP1, PSP2, PSF, and p54nrb; Fox et al, 2002Fox et al, , 2005Shav-Tal et al, 2005) relocalize in perinucleolar caps upon RNA polymerase II inhibition where they can be found in the concave caps (Shav-Tal et al, 2005).…”

“…These experiments showed that only CF I m 68 and PSP1 were present in the same perinucleolar cap structures upon inhibition of transcription. This result is consistent with the observation that all other paraspeckle proteins known so far (PSP1, PSP2, PSF, and p54nrb; Fox et al, 2002Fox et al, , 2005Shav-Tal et al, 2005) relocalize in perinucleolar caps upon RNA polymerase II inhibition where they can be found in the concave caps (Shav-Tal et al, 2005).Nuclear speckles correspond to PFs and IGCs at the electron microscopy level. PFs are fibrillar structures measuring 3-5 nm in diameter, and they are found distributed throughout the nucleoplasm and also at the periphery of IGCs (Monneron and Bernhard, 1969).…”

“…After inhibition of transcription, nuclear speckles become more prominent (Carmo-Fonseca et al, 1992;Spector et al, 1993), whereas paraspeckles' markers, such as PSP1 or PSF, relocalize to perinucleolar caps Shav-Tal et al, 2005). We therefore went on to determine the effect of transcription inhibition on CF I m 68 localization.…”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…These stresses cause condensation and segregation of the fibrillar centre and the granular component. This is accompanied by the formation of 'caps' of nucleoplasmic RNA-binding proteins on the body of nucleoli (Boulon et al, 2010;Shav-Tal et al, 2005). It has been reported that nucleolar capping is observed in HCMV-infected cells (Gaddy et al, 2010).…”

“…These stresses cause condensation and segregation of the fibrillar centre and the granular component. This is accompanied by the formation of 'caps' of nucleoplasmic RNA-binding proteins on the body of nucleoli (Boulon et al, 2010;Shav-Tal et al, 2005 et al, 2012c). It is unknown whether HCMV infection stimulates p53-mediated nucleolar stress responses, which include specific changes to the protein translational profile of the cell (Boulon et al, 2010).…”