Effects of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis

Abstract

During mitosis some nuclear complexes are relocalized at the chromosome periphery and are then reintegrated into the re-forming nuclei in late telophase. To address questions concerning translocation from the chromosome periphery to nuclei, the dynamics of one nucleolar perichromosomal protein which is involved in the ribosomal RNA processing machinery, fibrillarin, was followed. In the same cells, the onset of the RNA polymerase I (RNA pol I) activity and translocation of fibrillarin were simultaneously investigated. In PtK1 cells, RNA pol I transcription was first detected at anaphase B. At the same mitotic stage, fibrillarin formed foci of increasing size around the chromosomes, these foci then gathered into prenucleolar bodies (PNBs) and later PNBs were targeted into the newly formed nucleoli. Electron microscopy studies enabled the visualization of the PNBs forming the dense fibrillar component (DFC) of new nucleoli. Anti-fibrillarin antibodies microinjected at different periods of mitosis blocked fibrillarin translocation at different steps, i.e. the formation of large foci, foci gathering in PNBs or PNB targeting into nucleoli, and thereby modified the ultrastructural organization of the nucleoli as well as of the PNBs. In addition, antibody-bound fibrillarin seemed localized with blocks of condensed chromatin in early G1 nuclei. It has been found that blocking fibrillarin translocation reduced or inhibited RNA pol I transcription. It is postulated that when translocation of proteins belonging to the processing machinery is inhibited or diminished, a negative feed-back effect is induced on nucleolar reassembly and transcriptional activity.

Real-time imaging of RNA polymerase I activity in living human cells

“…Nucleolar architecture undergoes extensive changes throughout the mitosis, during which rDNA transcription is repressed and reactivated ( Nemeth and Grummt, 2018 ; Weisenberger and Scheer, 1995 ; Zharskaia and Zatsepina, 2007 ). Using FISH or BrUTP incorporation to detect pre-rRNAs in fixed cells, it has been estimated that rRNA synthesis generally pauses in metaphase and restarts at late anaphase or early telophase ( Fomproix et al, 1998 ; Hernandez-Verdun et al, 2002 ; Leung et al, 2004 ; Morcillo et al, 1976 ; Roussel et al, 1996 ). However, there are no appropriate methods for analyzing spatiotemporal dynamics of rRNA synthesis throughout mitosis by tracing single cells.…”

Integrating Wheat Nucleolus Structure and Function: Variation in the Wheat Ribosomal RNA and Protein Genes

“…The TATA box regions are emphasized in red and are postulated to engage the TATA box-binding protein (TBP) for contributing to engaging RNA polymerase II in forming an R-loop shield. (C) Model for the protein complexes formed in the 26S gene-3 downstream region to ensure RNA pol I transcription is properly terminated, based on the studies on human rDNA structure and function (Fomproix et al, 1998;Abraham et al, 2020;doi.org/10.1038/s41586-020-2497. The prominent nucleolar proteins, nucleolin and fibrillarin, are possibly involved as discussed in the earlier text (Nasirudin et al, 2004;Pontvianne et al, 2007;Rodriguez-Corona et al, 2015).…”

“…Major nucleolus proteins, such as the nucleolin and fibrillarin gene models in wheat (Supplementary Figure 1), have RNAinteracting domains that could also contribute to facilitating the establishment of a proposed R-loop shield. Rodriguez-Corona et al (2015) noted that the rDNA transcription instability in permeabilized mammalian tissue culture cells infused with fibrillarin antibodies (Fomproix et al, 1998) may be due to the antibodies preventing fibrillarin from contributing to the R-loop shield, which normally blocks sense sincRNA formation by RNA pol I (Figure 2).…”

Spatial organization and dynamics of chromosomes and microtubules during barley mitosis

“…The chromosome organization and dynamics are controlled by the activity of structural maintenance of chromosomes (SMC) complexes. The chromosomes condense by the activity of Condensin I and II complexes into the rodlike structures with sister chromatids being held together along the arms and particularly in centromeric regions by the Cohesin complex (Fomproix et al., 1998; Gibcus et al., 2018; Hernandez‐Verdun, 2011). Nucleoli disperse at the beginning of mitosis and reemerge in course of telophase around 45S rDNA loci (Hernandez‐Verdun, 2011; Perutka et al., 2021).…”