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Bio 405/505 Cell Nucleus Lectures Session 5 (4/16/09) The Nucleolus: rRNA Gene Organization and Function 1) Koberna et al., J Cell Biol 157 (2002) 743-748.

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Presentation on theme: "Bio 405/505 Cell Nucleus Lectures Session 5 (4/16/09) The Nucleolus: rRNA Gene Organization and Function 1) Koberna et al., J Cell Biol 157 (2002) 743-748."— Presentation transcript:

1 Bio 405/505 Cell Nucleus Lectures Session 5 (4/16/09) The Nucleolus: rRNA Gene Organization and Function 1) Koberna et al., J Cell Biol 157 (2002) 743-748. 2) Raska, Trends Cell Biol 13 (2003) 517-525.

2 Nucleolus The Nucleolus (‘tiny nucleus”) is a reticular fibrogranular structure in the nucleus that is specialized for transcription of ribosomal RNA and its packaging into pre-ribosomal subunits. The genes for ribosomal RNA are highly amplified and located on five different chromosomes called the nucleolar organizer regions (NOR). rDNA genes are concentrated within the numerous fibrillar centers (fc) that compose the nucleolus. rRNA is believed to be transcribed at the borders of fc and the dense fibrillar component (dfc) where ribosomal proteins associate to form pre-ribsosomal RNP particles but this is still controversial. Progressive processing of the pre–rRNP particles occurs within the granular component (gc) where mature ribosomal subunits are released for transport into the cytoplasm. The massive level of rRNA transcription is illustrated by the “Christmas tree” structures composed of repeating nascent rRNP strands that grow in assembly line fashion along the rDNA transcription units.

3 Raska, TRENDS in Cell Biology 2003, Figure 1. Schematic representation of human ribosomal RNA (rRNA) gene-array, rRNA gene and rRNA gene transcript. Schematic representation of ribosome

4 Mammalian Cell as Seen by Light Microscope (courtesy to lamondlab.com)

5 Nuclear Transcription in Mammalian Cells The RNA transcription signal is present in numerous sites throughout nucleus. The strongest signal is accumulated within nucleoli. Nucleoli are sites of expression of specific genes: ribosomal genes (rDNA)

6 Chromosome 13 Chromosome 14 Chromosome 15 The ribosomal RNA genes are highly amplified (total of ~400 copies in human cells) at the p-arms (Nucleolar Organizer Regions) of these 5 human NOR-chromosomes

7 Courtesy to http://www.dundee.ac.uk Visualization of Ribosomal Genes on Metaphase Chromosomes

8 Controversy behind the localization of nucleolar transcription sites Transcription sites are located exclusively in FCs Thiry et al; RNA. 2000, 12:1750-61. Thiry et al; Trends Cell Biol. 2001, 4:147 Cheutin et al; J Cell Sci. 2002, 115: 3297-307. Mais et al; J Struct Biol. 2002, 140: 214-26. Derenzini et al; J Histochem Cytochem. 2006, 54:131-45. Transcription sites are located exclusively in DFCs Melcak I et al; J Struct Biol. 1996, 116: 253-63. Cmarko et al; Histochem Cell Biol. 2000, 113:181-7. Gonzalez-Melendi et al; Plant J. 2001, 3: 223-33. Stanek D et al; Chromosoma. 2001, 110: 460-70. Koberna et al; J Cell Biol. 2002 157: 743-8.

9 Koberna et al., J Cell Biol. 2002;157(5):743-8. Ribosomal Genes in Focus: New Transcripts Label the Dense Fibrillar Components and Form Clusters Indicative of "Christmas Trees" in situ.

10 Major Conclusions of Koberna et al., 2002 1)At the fluorescence microscopic level, ribosomal genes are organized in discrete foci. # of foci varied from 10 to 40 in different cells (Fig 1 & 2). 2)rDNA transcription sites are strictly limited to nucleolar DFC and DFC/FC transition zones. This suggests a strict structure- function compartmentalization of the nucleolus (Fig 3). 3) (Correlation of fluorescence and electron microscopy) Each fluorescent nucleolar transcription site (focus) at the ultrastructural level corresponds to group of rDNA genes located within DFC that surround individual FC. Since in HeLa cells ~ 150 of ribosomal genes are transcriptionally active (Jackson et al., 1993; EMBO J; 12: 1059-1065) each focus represents a group in average of ~4 to15 active genes (Fig 1- 4).

11 Conclusion 1 (Fig 1 & 2) In HeLa cells ribosomal genes are organized in discrete groups or foci. Number of foci varied from 10 to 40 in different cells.

12 Koberna et al JCB 2002, Figure 1. Localization of ribosomal genes in HeLa cells. Fluorescence mapping of ribosomal genes (A and C, yellow), phase contrast image (B and C, blue), and the merge image (C) are shown. Ribosomal genes are located in a number of fluorescent foci. Most of the foci are situated in the nucleolus. Two foci are situated in the perinucleolar chromatin (arrowheads). (D) EM mapping of ribosomal genes. The signal (arrowheads) is located in DFC. (f, d and g) FCs, DFCs, and granular components, respectively Bars: (A–C) 10  ; (D) 200 nm.

13 (C–E) Light microscopic image of ribosomal genes (C and E, red) and transcription sites (D and E, green). The merged image is E. Most of rDNA foci exhibit the transcription activity. An rDNA focus without transcription signal is indicated by the arrow. Bar: 5  m. Koberna et al JCB 2002, Figure 2. LM mapping of nucleolar transcription in HeLa cells. High magnification is used to see details of nucleolar structure Ribosomal GenesTranscription Merged

14 Conclusion 2 rDNA transcription sites are strictly limited to nucleolar DFC and DFC/FC transition zones. This suggests a strict structure functional compartmentalization of nucleolus (Fig 3).

15 Koberna et al JCB 2002, Figure 3. EM mapping of nucleolar transcription in HeLa cells. (A) Nucleolar EM transcription signal observed in hypotonically treated cells is confined to the DFC. (B–D) EM localization of transcription in thin-sectioned permeabilized cell. (C and D) Detailed parts of B. Nucleolar transcription signal consists of numerous clusters of gold particles accumulated in the DFC.

16 Conclusion 3 (Correlation of fluorescence and electron microscopy) Each fluorescent nucleolar transcription site on ultrastructural level corresponds to group of rDNA genes located within DFC that surround individual FC. Since in HeLa cells ~ 150 of ribosomal genes are transcriptionally active (Jackson et al., 1993; EMBO J; 12: 1059-1065) each focus represents a group in average of ~4 to 15 active genes (Fig 1-4).

17 Immunolabeling tools for Light and Electron Microscopy Raska, TRENDS in Cell Biology 2003, Box 1 FLM EM FLM

18 Concomitant LM and EM Mapping of Transcription Signal in a Thin Section of a Permeabilized Cell. (B) Detailed part of A. The colored insert in A represents the wide- field LM picture of the same area depicting newly synthesized RNA (red) and DNA counterstained with YOYO (green). (C) Merge of an inverse EM image from A in cyan, and a transcription LM signal from the upper insert in red. Note the overlap of the LM and EM transcription signals (white areas). Bars: (A) 0.5  ; (B) 0.2 . Koberna et al JCB 2002, Figure 4.

19 Proposed model of the arrangement of Christmas tree structures in animal cells. This model suggests that FC represent sites of storage/ reassembly of the rDNA transcription machinery as well as inactive rDNA. DFC represent sites of rRNA synthesis/processing, while in GC the final steps take place for rRNA processing and assembly of pre-ribosomal particles. Raska, TRENDS in Cell Biology 2003, Figure 5

20 Major Conclusions of Koberna et al., 2002 1)In HeLa cells ribosomal genes are organized in discrete groups or foci. Number of foci varied from 10 to 40 in different cells (Fig 1 & 2). 2)rDNA transcription sites are strictly limited to nucleolar DFC and DFC/FC transition zones. This suggests a strict structure functional compartmentalization of nucleolus (Fig 3). 3) (Correlation of fluorescence and electron microscopy) Each fluorescent nucleolar transcription site on ultrastructural level corresponds to group of rDNA genes located within DFC that surround individual FC. Since in HeLa cells ~ 150 of ribosomal genes are transcriptionally active (Jackson et al., 1993; EMBO J; 12: 1059-1065) each focus represents a group in average of ~4 to15 active genes (Fig1- 4).

21 Kalmárová et. al., J Struct Biol. 2007;160(1):49-56. Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli.

22 Representation of chromosome territories in the fibroblast nuclei Bolzer et al., PLoS Biol. 2005

23 Major Conclusions of Kalmárová et al., 2007 1)Majority of the NOR bearing chromosome territories are spatially associated with the nucleolus (Fig 1 and Fig 3). 2) NORs are usually located within the nucleolus regardless of their transcription activity (Fig 4) 3) NOR bearing chromosome territories which are not associated with the nucleolus may loop out rDNA sequences into the nucleolus (Fig.2 and Fig 5)

24 Conclusion 1 (Fig 1 and 3) Majority of the NOR bearing chromosome territories are spatially associated with the nucleolus

25 Kalmárová et. al., Fig. 1. Association of the chromosomes 13 and 15 with nucleoli in interphase HeLa (a) and LEP (b) cells. The transformed HeLa cells contain three homologs of the chromosomes 13 and four homologs of the chromosome 15. The primary LEP cells have normal karyotype with two chromosomes 13 and 15. In both HeLa and LEP cells, the chromosome homologs 15 appeared usually in close association with nucleoli, while one of the homologs 13 was often distanced from the nucleoli (arrow).

26 Fig. 3. Transcription competence of the NORs belonging to different chromosomes correlates with nucleolar associations of these chromosomes. The percentage of the nucleoli closely associated chromosomes was counted in HeLa and LEP cells.

27 Conclusion 2 (Fig 4) NORs are usually located within the nucleolus regardless of their transcription activity

28 Fig. 4. Most HeLa and LEP cells contain no extranucleolar rDNA. (a) rDNA (red), fibrillarin (green) and merged image in a HeLa cell. No rDNA signals are present outside the fibrillarin-positive nucleoli. The arrows indicate Cajal bodies. Bar: 10 lm. (b) The percentage of extranucleolar rDNA foci in HeLa and LEP cells. The extranucleolar rDNA foci are absent in about 73% of HeLa cells and in about 87% of LEP cells.

29 Conclusion 3 (Fig.2 and Fig. 5) NOR bearing chromosome territories which are not associated with the nucleolus may loop out rDNA sequences into the nucleolus

30 Kalmárová et. al., Fig. 2. A thin protrusion between the extranucleolar chromosome territory and nucleolus. Two chromosome homologs 14 (in red; arrows) in the HeLa cell are closely associated with fibrillarin-positive nucleoli (in green). The third chromosome homolog 14 (in red) is distanced from nucleolus and connected to it via a long chromatin protrusion that ends with a thickening at the contact point with the nucleolus.

31 Fig. 5. Triple labeling of chromosomes 14, fibrillarin and rDNA in a HeLa cell. The chromosomes are shown in blue, fibrillarin in green, and rDNA in red. Two chromosome territories are closely associated with nucleoli (arrowheads). The third chromosome territory (arrow) is distanced from the nucleolus and is not colocalized with rDNA signal. Since no rDNA signals were found outside the nucleoli, the NOR belonging to this chromosome should be in the nucleolus. Bar: 10 lm.

32 Major Conclusions of Kalmárová et al., 2007 1)Majority of the NOR bearing chromosome territories are spatially associated with the nucleolus (Fig 1 and Fig 3). 2) NORs are usually located within the nucleolus regardless of their transcription activity (Fig 4) 3) NOR bearing chromosome territories which are not associated with the nucleolus may loop out rDNA sequences into the nucleolus (Fig.2 and Fig 5)


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