lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperone–like mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production.

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 Thr131 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.

SUMO-1 Modification Alters ADAR1 Editing Activity

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

Building an efficient factory

The subnucleolar structure that is involved in rDNA transcription has been controversial. A report by Koberna et al. (2002)(this issue, page 743) adds significant weight toward the idea that dense fibrillar components (DFCs)**Abbreviations used in this paper: DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; Pol I, polymerase I. and fibrillar center (FC)/DFC borders are the sites of pre-rRNA synthesis.

Revealing the unseen: the organizer region of the nucleolus

We carried out a high-resolution ultrastructural analysis of the nucleolus in mouse P815 cells by combining specific DNA and RNA staining, anti-fibrillarin immunolabeling, contrast enhancement by energy filtering TEM and phosphorus mapping by ESI to visualize nucleic acids. We demonstrated that specifically contrasted DNA, fibrillarin and phosphorus overlap within the nucleolar dense fibrillar component. Moreover, we describe a ‘DNA cloud’ consisting of an inner core of DNA fibers (fibrillar center) and a periphery made of extremely thin fibrils overlapping the anti-fibrillarin immunolabeling (dense fibrillar component). This highly sensitive approach has allowed us to demonstrate, for the first time, the exact distribution of DNA within the decondensed interphase counterpart of the NOR, which includes both the fibrillar center and the dense fibrillar component.

Localization of DNA in the fibrillar components of the nucleolus: a cytochemical and morphometric study.

We studied the distribution of DNA in human circulating lymphocyte nucleoli using three different cytochemical methods for selective visualization of DNA in thin sections: the Feulgen-like osmium-ammine reaction, the NAMA-Ur procedure, and the osmium-ammine staining in glycine buffer, pH 1.5. All three methods indicated the presence of uniformly distributed, highly decondensed DNA filaments forming a large solitary agglomerate in the central part of the nucleolar area, corresponding to the solitary large fibrillar center (FC) as revealed by uranium and lead staining. We also studied the relationship between DNA agglomerates and nucleolar fibrillar components in resting and phytohemagglutinin (PHA)-stimulated lymphocytes by morphometric analysis of the areas occupied by these structures. In resting lymphocytes the mean area of the DNA agglomerates was 0.479 micron 2 +/- 0.161 SD, whereas that of FCs was 0.380 micron 2 +/- 0.149 SD, with a ratio of 1.26. In PHA-stimulated lymphocytes the mean area of the DNA agglomerates was 0.116 micron 2 +/- 0.056 SD, whereas that of the FCs was 0.075 micron 2 +/- 0.032 SD, with a ratio of 1.55. In PHA-stimulated lymphocytes we also measured the area occupied by the FCs plus the closely associated dense fibrillar component (DFC). The mean value of these two fibrillar components was 0.206 micron 2 +/- 0.081 SD. These data demonstrate that decondensed DNA filaments are uniformly distributed in the FCs and that in transcriptionally active nucleoli they are also present in the proximal portion of the DFC surrounding the FCs.

Ultrastructure and the relationship between the nucleolus and the nucleolar organizer region in Orthoptera male germ cells

The ultrastructure of the nucleolus and the nucleolar organizer region, together with the relationships they maintain, are described in male germ cells of two species of grasshoppers. The nucleolus is multiple and appears related to a fibrillar center which seems to represent only a nontranscribing part of the organizer region. By means of serial sections, a particular relationship among the fibrillar center, the rDNA loops, and the nucleolar masses is suggested.Key words: ultrastructure, nucleolar organizers, nucleolus, Orthoptera.