20 research outputs found
Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits
Subsets of 40S ribosomal subunits are required for initiating rRNA processing, rRNA maturation, and nuclear export
Pre-Ribosomal RNA Processing in Human Cells: From Mechanisms to Congenital Diseases
Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases
The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis
The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed
The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA
RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation
4D visualization of UBF-GFP dynamics during inhibition of rRNA synthesis in the same living KB cells as presented on Fig 2.
<p>(AâT) An example of 4D dynamics demonstrating successive fusion and gathering of five UBF positive spheroids (N°1 âN°5) contained in a ROI (delimited by the blue dotted line). Between 2 h 45 min and 7 h 30 min, we noticed the following steps: spheroids N°1and N°2 fused to give structure A; the latter gathered with N°3 to give B, and N°4 and N°5 finally gathered to B to give C. Note that the cap resulting from the fusion and gathering of the different spheroids is more compact than the initial region limited by the blue dotted line. The scale bar represents 2 ÎŒm.</p
4D dynamics of UBF-GFP during inhibition of rRNA synthesis in living KB cells.
<p>(AâD), gallery of fluorescence maximum intensity projections and 3D reconstructions displaying the dynamics of UBF-GFP during nucleolar segregation. Reorganization of UBF-GFP spots was based on their successive gathering, fusion, enlargement, and final grouping into two or three caps localized at the nucleolar periphery. Blue and red arrowheads identify two large nucleoli to demonstrate their rotation within the nucleus during the step-by-step reorganization of UBF-GFP spots and (E1-E4) their 3D visualization at times 0 and 8 h respectively. The scale bars represent 10 ÎŒm.</p
Ultrastructural pre-embedding localization of UBF within the nucleolus of control KB cells.
<p>(A) An abundant labeling (silver/gold particles) inside several fibrillar centers (fc) (outlined by a white dotted line). A few particles (black arrows) were visible within the nucleolar dense fibrillary component (dfc), nucleolar granular component (gc), nucleoplasm, or cytoplasm. (B, C) At high magnification, numerous particles were located within fibrillar centers (fc) (outlined by white dotted lines). The dfc is outlined by a black line on C. The scale bars represent 500 nm.</p
Spatial proximity of intranucleolar chromatin (ICC), perinucleolar chromatin (PCC), and UBF spots in fixed control cells.
<p>(A) 3D reconstruction of the nucleus in a HeLa cell stably expressing histone H2B-GFP using volume rendering. A virtual cube inserted into the nuclear volume delineates the nucleolar territory (nl) extracted in order to analyze its interior. (B) The nucleolar volume reconstructed using simultaneous volume rendering and surface visualization of intranucleolar H2B-GFP for better visualization of ICC. After volume extraction, the nucleolar interior revealed a network of ICC interconnected with the PCC shell. (C, D) Relationships between ICC (green) and UBF spots (red) within the nucleolus of KB cells cotransfected with H2B-GFP and UBF-dsRed. These 3D views demonstrate, inside the nucleolus, the presence of the ICC network in contact with UBF spots. The scale bar represents 3.5 ÎŒm in (A, B); 5 ÎŒm in (C) and 3.5 ÎŒm in (D).</p
The dynamics of histone H2B-GFP during inhibition of rRNA synthesis in living HeLa cells.
<p>This gallery of optical sections was extracted from z-stacks of corresponding time series and displayed with 15â45 min intervals to fix the main stages of ICC evolution (corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187977#pone.0187977.s025" target="_blank">S8 Movie</a>). The nuclei of three cells in the white rectangle on the initial image (âStart of experimentâ) were enlarged to visualize the evolution of the ICC in more detail. In the course of AMD treatment we observed a gradual condensation of filamentous structures of the ICC into coarse clumps during 120 min with their migration from the nucleolar interior towards the PCC shell (165 min). During this movement several ICC clumps approached each other and fused just before the coalescence with the PCC. Note that at the end of the experiment (180 min) there was no more chromatin within the nucleolus. The scale bars represent 12 ÎŒm.</p
Interaction between UBF spots, ICC, and PCC within the pre-segregated nucleoli of HeLa and KB cells.
<p>(A-C) Consecutive optical sections (S.21-23) of nucleoli in HeLa cells stably expressing histone H2B-GFP. Note the prominent ICC clumps within the nucleolar interior after rRNA synthesis inhibition. These cells were immunolabeled for UBF at the end of time-lapse imaging (the corresponding nuclei are marked by red stars on A, D). (D-F) The same optical sections merged with 3D surface rendering of UBF spots (red). Note the direct contact between UBF spots and ICC inclusions. At the same time UBF spots that shifted to the nucleolar periphery became incorporated into the solid PCC shell. (G-I) 3D reconstructions of enlarged UBF spots extracted from the nucleolus of a KB cell doubly transfected with H2B-GFP and UBF-dsRed plasmids, fixed and imaged. A typical pre-segregated nucleolus containing five large UBF spots reconstructed using volume rendering (green) and surface visualization (red). Incorporation of peripherally-located UBF spots into the massive PCC shell as well as their contact with ICC can be readily recognized. Note the ICC clump that is in contact with two UBF spots. After extraction according to the virtual cubes in H, images were rotated at an appropriate angle to exemplify how UBF spots can be linked to each other by ICC. The scale bars represent 10 ÎŒm in (A); 5 ÎŒm in (G, H); 2 ÎŒm in (I).</p