The nucleolar architecture of polymerase I transcription and processing.

Abstract

The nucleolus, the site of transcription and processing of the major ribosomal genes, generally reveals three distinct ultrastructural components in conventional thin‐section electron micrographs (fibrillar centres, dense fibrillar component and granular component). We show here that different parts of the transcription and transcript processing pathway can be mapped to the different nucleolar components in pea root cells. This study shows the full three‐dimensional arrangement of the different domains by in situ hybridization and confocal microscopy, and their correspondence with the major ultrastructural components of the nucleolus is revealed by parallel serial section electron microscopy. The active rDNA is widely dispersed in discrete foci, the larger of which, at least, correspond to well‐defined fibrillar centres. A probe to the external transcribed spacer (ETS) sequence of the pre‐rRNA transcripts labels clearly demarcated regions surrounding the foci of rDNA, and which we show correspond to the dense fibrillar component. Finally, a probe to the entire 45S transcript shows a higher concentration in regions corresponding to the granular component, surrounding the dense fibrillar component labelled by the ETS probe. The changes in structure that occur with heat shock show that nucleolar organization is dynamic and dependent upon transcriptional activity. These results show that the various RNA processing events are spatially highly organized and suggest a vectorial or radial model of transcription and transcript processing, where nascent and newly completed transcripts occupy zones surrounding the genes, which are in turn surrounded by regions containing the older more mature transcripts.

Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

“…Clustered genes are associated with the fibrillar centers in the nucleoli (Junera et al, 1995;Shaw et al, 1995), which has been proposed to be attachment sites on the matrix (Hozak et al, 1994). It seems that the process of transcription is associated with dispersion of the rDNA from these attachment sites.…”

“…A complementary approach has been to correlate this 3-D organization with the nucleolar domains defined at the level of electron microscopy. The clusters of genes were localized in the fibrillar centers of the nucleoli (Junera et al, 1995;Shaw et al, 1995) with a radial organization of the early transcripts in the dense fibrillar component (Shaw et al, 1995).…”

“…The 3-D distribution of the rDNA in nucleoli appeared heterogeneous, and it was proposed that this heterogeneity depends on the transcriptional activity of the rDNA Shaw, 1989, 1990;Leitch et al, 1992;Highett et al, 1993;Robert-Fortel et al, 1993;Junera et al, 1995;Shaw et al, 1995). Therefore, the distribution of the rDNA transcription machinery would be very important to confirm this hypothesis and to know whether transcription could also be dependent or modulated by the 3-D distribution of the transcription factors.…”

Histone H4 Acetylation of Euchromatin and Heterochromatin Is Cell Cycle Dependent and Correlated with Replication Rather Than with Transcription

“…Only a subset of rRNA genes is actively transcribed during interphase (Shaw et al, 1995), even in yeast with comparatively few rRNA genes (Dammann et al, 1993), and heavily transcribed cistrons are not organized in nucleosomal structures (e.g., Sogo et al, 1984; Conconi et al, 1992; Dammann et al, 1993). Apparently, transcriptional activity within nucleoli is upregulated by increased activity of already active cistrons that are free of nucleosomes (reflected by "Christmas tree"-like structures of nascent transcripts in electron microscopic images; Miller and Beatty, 1969) rather than by activation of …”

Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes

“…Examples include global effects like chromosome territories (Foster and Bridger 2005;Cremer et al 2006) and transcription factories (Faro-Trindade and Cook 2006), and specific effects such as telomeres and centromeres at the yeast nuclear periphery (Heun et al 2001a;Hediger et al 2002), the large ribosomal DNA repeats at the nucleoli of all organisms (Shaw et al 1995), and the 5S rDNA at or near the nucleoli of many organisms . Multiple genes alter their position when transcriptionally activated (Buchenau et al 1997;Brown et al 1999;Volpi et al 2000;Kosak et al 2002;Casolari et al 2004Casolari et al , 2005Harnicarova et al 2006), and some genes are brought together with linearly distant enhancer elements (Liu and Garrard 2005;Spilianakis et al 2005;Liu and Francke 2006;Su et al 2006).…”