Transcript Powerpoint Slides - Department of Biological Sciences

Bio 525/ Spring, 2010
Nuclear Architecture and Genomic
Function
Session 6, Part II: The Nucleolus: rRNA
Gene Organization and Function:
Background and Figures for Koberna et
al.,2002; Kalmárová et al., 2007
• 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.
Nucleolus
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
The Nobel Prize in Chemistry in 2009
was awarded to Venkatraman
Ramakrishnan, Thomas A. Steitz and Ada
E. Yonath for having showed what the
ribosome looks like and how it functions
at the atomic level.
A Beautiful Complexity of the
Ribosomal Structure
Nissen et al., Science : Vol. 289, pp. 920 - 930
Mammalian Cell as Seen by Light Microscope
(courtesy to lamondlab.com)
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)
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
Visualization of Ribosomal Genes on Metaphase Chromosomes
Courtesy to http://www.dundee.ac.uk
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.
Ribosomal Genes in Focus: New
Transcripts Label the Dense Fibrillar
Components and Form Clusters Indicative
of "Christmas Trees" in situ.
Koberna et al., J Cell Biol. 2002;157(5):743-8.
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).
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.
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 m; (D) 200 nm.
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 Genes
Transcription
Merged
(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 mm.
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).
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.
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).
Raska, TRENDS in Cell Biology 2003, Box 1
Immunolabeling tools for Light and Electron Microscopy
Koberna et al JCB 2002, Figure 4.
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 widefield 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 m; (B) 0.2 m.
Raska, TRENDS in Cell Biology 2003, Figure 5
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.
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).
Spatio-temporal dynamics at rDNA foci:
Global switching between DNA replication
and transcription
Pliss et al., J Cell Bichem. 2005; 94: 554-565
Major Conclusion of Pliss et al., 2005
The rDNA foci are the functional units for coordinating
replication and transcription of the rRNA genes in space
and time. This involves a global switching mechanism,
active from mid to late S-phase, for turning off
transcription and turning on replication at individual
rDNA foci. Once all the rRNA genes at individual foci
are replicated, these higher order chromatin domains
are reprogrammed for transcription.
Figure 2. Electron microscopic
localization of replication, transcription,
and fibrillarin in HeLa cells.
A: Electron microscopic localization of
transcription in thin sectioned cells. B:
Fluorescence microscopy image of
nucleolar transcription pattern seen in
cells synchronized in S-phase. C:
Enlarged outlined area in (A). D: The
nucleolar DNA replication: the signal is
found mainly in the DFC (arrows). Some
DNA replication occurs also in the
perinucleolar chromatin (arrowheads). E:
Fibrillarin –a marker of the rRNA
processing is accumulated in the DFC.
Figure 1. Imaging of rDNA, nucleolar transcription, replication, and fibrillarin in HeLa cells.
A-D: Mapping of rDNAs (A, green), transcription (B, red), and fibrillarin (C, blue); (D) merged image. Most rDNA
foci co-localize with transcription signal and fibrillarin. Two rDNA foci (arrows) do not overlap with transcription and
fibrillarin signals. E-G: Signals of replication (E, green) and rDNAs (F, red). One rDNA focus (arrow) overlaps with
replication signal at the merged image (G). H-K: Mapping of replication (H, green), rDNAs (I, red), and
transcription (J, blue) in the nucleolus. The rDNA focus, which co-localizes with replication signal (arrow), does not
overlap with transcription in the merged image (K). Foci inactive in both DNA replication and transcription are
occasionally observed (see arrowhead in K). L-O: Mapping of replication (L, green), rDNAs (M, red), and fibrillarin
(N, blue) in the nucleolus. In the merged image (O), no fibrillarin signal is seen in the replicating rDNA foci
(arrows), while magenta colored sites (arrowheads) co-localize with fibrillarin and do not replicate DNA .
Figure 3. Schematical representation of rDNA foci dynamically switched on and off for DNA
replication or transcription.
In this model, individual rDNA foci are depicted as being along the borders of the nucleolar FC/DFC regions. Each
focus is a functional unit containing multiple rDNA genes switched on for either DNA replication (green) or
transcription (red). Following the completion of DNA replication at individual foci, transcriptional activity is switched
back on.
Major Conclusion of Pliss et al., 2005
The rDNA foci are the functional units for coordinating
replication and transcription of the rRNA genes in space
and time. This involves a global switching mechanism,
active from mid to late S-phase, for turning off
transcription and turning on replication at individual
rDNA foci. Once all the rRNA genes at individual foci
are replicated, these higher order chromatin domains
are reprogrammed for transcription.
Positioning of NORs and NOR-bearing
chromosomes in relation to nucleoli.
Kalmárová et. al., J Struct Biol.
2007;160(1):49-56.
Representation of
chromosome
territories in the
fibroblast nuclei
Bolzer et al., PLoS Biol.
2005
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)
Conclusion 1 (Fig 1 and 3)
Majority of the NOR bearing chromosome
territories are spatially associated with
the nucleolus
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).
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.
Conclusion 2 (Fig 4)
NORs are usually located within the
nucleolus regardless of their
transcription activity
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.
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
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.
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.
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)