NUCLEOLUS AND IT’S NUCLEOLUS AND IT’S ORGANIZATIONORGANIZATION

EXPLORING THE NUCLEUS EXPLORING THE NUCLEUS INTERIOR…………..INTERIOR…………..

CONTENTSCONTENTS

► What is a nucleolus?????What is a nucleolus?????► The milestones of early nucleolus research ….The milestones of early nucleolus research ….► Features of nucleolusFeatures of nucleolus► Morphology of nucleolusMorphology of nucleolus► Evolution of tripartite organizationEvolution of tripartite organization► Nucleolar organization and dynamicNucleolar organization and dynamic► Nucleolus in mitosisNucleolus in mitosis► The plurifunctional nucleolus► The Nucleolus and Human diseasesThe Nucleolus and Human diseases► ConclusionConclusion

What is a nucleolus?????What is a nucleolus?????

► A prominent sub-nuclear A prominent sub-nuclear structure that is not bound structure that is not bound by a membrane and resides by a membrane and resides within the nuclear matrix. within the nuclear matrix.

► The site of the biogenesis of The site of the biogenesis of ribosomal RNA (rRNA) and ribosomal RNA (rRNA) and assembly of ribosomes.assembly of ribosomes.

► Called as “Ribosome Called as “Ribosome Factories”Factories”

Source: Molecular Expressions: micro.magnet.fsu.edu/.../nucleolus

The milestones of early The milestones of early nucleolus research ….nucleolus research ….

► 1835-1835-Discovery of the nucleolus (termed "Keimfleck" or Discovery of the nucleolus (termed "Keimfleck" or "macula germinatova") in germinal vesicles"macula germinatova") in germinal vesicles

► 1839-1839-Introduction of the term "nucleolus" (small nucleus or Introduction of the term "nucleolus" (small nucleus or nucleus of the nucleus).nucleus of the nucleus).

► 1893-1893-Observation that the nucleoli gradually disappear Observation that the nucleoli gradually disappear during prophase.during prophase.

► 1934-1934-Demonstration of special chromosomal regions called Demonstration of special chromosomal regions called secondary chromosomal constrictions or nucleolus-organizing secondary chromosomal constrictions or nucleolus-organizing regions (NORs).regions (NORs).

► 1952-1952-First isolation of nucleoli.First isolation of nucleoli.► 1962-1962-After a pulse of radioactive precursor, the label is After a pulse of radioactive precursor, the label is

initially found in 45S RNA and then transferred from 45S to initially found in 45S RNA and then transferred from 45S to 28S RNA.28S RNA.

► 1969-1969-The spreading technique made it possible to visualize The spreading technique made it possible to visualize the transcription process. Each transcription unit looks like a the transcription process. Each transcription unit looks like a "Christmas tree". "Christmas tree".

Features of nucleolusFeatures of nucleolus

► Dark stainedDark stained body in the nucleus body in the nucleus

► Formed by the Formed by the nucleolar organizing regions (NOR)nucleolar organizing regions (NOR) of DNA of DNA

► A dynamic structure: A dynamic structure: Nucleoli are not staticNucleoli are not static structures. They structures. They disassemble during mitosis and reform in early G1 phase .disassemble during mitosis and reform in early G1 phase .

► Size and number vary according to the requirement of cell.Size and number vary according to the requirement of cell.

► A site for A site for ribosome biogenesis.ribosome biogenesis.

Morphology of nucleolusMorphology of nucleolus

Nucleoli have got tripartite Nucleoli have got tripartite organization composed of organization composed of three morphologically three morphologically distinct regions :-distinct regions :-

► Fibrillar Center (FC)Fibrillar Center (FC)

► Dense Fibrillar Center or Dense Fibrillar Center or Dense Fibrillar Component Dense Fibrillar Component (DFC)(DFC)

► Granular region or Granular region or Granular Component (GR)Granular Component (GR)

Fig: The FC(asterisks), the DFC(arrow) and GC(G) Bar: 0.5 μm (source: review-springerlink springer-Verlag 200510.1007/s00418-005-0046-4)

Fibrillar Center (FC)Fibrillar Center (FC)

► lightly stained when lightly stained when observed by EM observed by EM

► composed of "fibrils" (± composed of "fibrils" (± 50Ǻ)50Ǻ)

► contains contains DNA that is not DNA that is not actively being transcribedactively being transcribed

► presence of pol Ipresence of pol I► multiple FC in one nucleolus multiple FC in one nucleolus ► their number doubles in G2 their number doubles in G2

compared to G1 compared to G1 ► accounts for only 1-2 accounts for only 1-2

percent of the total volume percent of the total volume of the nucleolus of the nucleolus

Dense Fibrillar CenterDense Fibrillar Center (DFC) (DFC)

► Surround the FC’s Surround the FC’s ► Composed of "densely Composed of "densely

packed fibrils" (30-50 Ǻ) packed fibrils" (30-50 Ǻ) ► Contains Contains RNA molecules RNA molecules

being transcribed being transcribed ► Occupies a large fraction of Occupies a large fraction of

the nucleolus, ± 17 percent.the nucleolus, ± 17 percent.

Fig: The dense fibrillar component (arrow) and the granular component (G). Bar: 0.3 μm

(source: review-springerlink springer-Verlag 200510.1007/s00418-005-0046-4)

Granular regionGranular region (GR) (GR)

► Region encompassing both Region encompassing both the FC and the DFC the FC and the DFC

► Consisting of granules 150-Consisting of granules 150-200 Ǻ 200 Ǻ

► Contains Contains maturing ribosomal maturing ribosomal precursorprecursor particles particles

► Granule rich region due to the Granule rich region due to the presence of RNP particles presence of RNP particles

► With a fraction of about 75 With a fraction of about 75 percent, it occupies the percent, it occupies the largest fraction of the total largest fraction of the total nucleolus volume nucleolus volume

► It separates nucleolus and It separates nucleolus and nucleoplasmnucleoplasm

Fig: The dense fibrillar component (arrow) and the granular component (G). Bar: 0.3 μm (source: review-springerlink springer-Verlag 200510.1007/s00418-005-0046-4)

Evolution of tripartite Evolution of tripartite organizationorganization

► The The "tripartite’ organization""tripartite’ organization" (FC, DFC, GC) of the nucleolus is (FC, DFC, GC) of the nucleolus is

commonly accepted.commonly accepted.► Proposed that this particular organization is only observed in Proposed that this particular organization is only observed in

higher eukaryoteshigher eukaryotes ► It It evolved fromevolved from a bipartite organization with the transition a bipartite organization with the transition

from from anamniotes to amniotesanamniotes to amniotes..► Reflecting the substantial increase in the rDNA intergenic Reflecting the substantial increase in the rDNA intergenic

region, an region, an original fibrillar componentoriginal fibrillar component would have separated would have separated into the FC and the DFCinto the FC and the DFC (Thiry and Lafontaine 2005). (Thiry and Lafontaine 2005).

Other components of NucleolusOther components of Nucleolus

► A substantial (additional) component of the nucleolus is A substantial (additional) component of the nucleolus is chromatinchromatin, which penetrates the organelle from the , which penetrates the organelle from the surrounding nucleoplasm.surrounding nucleoplasm.

► One last structure identified within the nucleolus is One last structure identified within the nucleolus is nucleolar nucleolar vacuolevacuole. There are multiple nucleolar vacuoles in the . There are multiple nucleolar vacuoles in the nucleolus, but it remains unclear whether or not they serve nucleolus, but it remains unclear whether or not they serve some functional or structural purpose.some functional or structural purpose.

► Several sub-nuclear compartments are often associated with Several sub-nuclear compartments are often associated with nucleoli including, the nucleoli including, the perinucleolar compartmentperinucleolar compartment (PNC); the (PNC); the Cajal Cajal and and Sam68 bodiesSam68 bodies; and ; and paraspeckles.paraspeckles.

Nucleolar organization and Nucleolar organization and dynamicsdynamics

► Transcription of ribosomal DNATranscription of ribosomal DNA (rDNA) by RNA polymerase I (rDNA) by RNA polymerase I occurs either in the occurs either in the fibrillar centers (FCsfibrillar centers (FCs) or at the boundary ) or at the boundary between the between the FCFC and and the dense fibrillarthe dense fibrillar componentcomponent (DFC) (DFC) region region

► Pre-ribosomal RNA transcripts are Pre-ribosomal RNA transcripts are spliced and modifiedspliced and modified by by small nucleolar ribonucleoproteins (snoRNPs) small nucleolar ribonucleoproteins (snoRNPs) in the DFCin the DFC. This . This is revealed by the presence of constituting proteins, for is revealed by the presence of constituting proteins, for example example fibrillarin, nucleolinfibrillarin, nucleolin..

► FinalFinal maturation maturation of the pre-ribosomal ribonucleoprotein and of the pre-ribosomal ribonucleoprotein and assembly with ribosomal proteins occurs mostly in the assembly with ribosomal proteins occurs mostly in the granular component (GC)granular component (GC) region. Protein B23 and NOP52 in region. Protein B23 and NOP52 in GC provide evidence for it.GC provide evidence for it.

Early prophase- cyclin B1–CDK1 levels increase, the transcription machinery usually remains attached to active NORs during mitosis, some RNA polymerase I subunits leave the fibrillar centre (FC).

Late prophase- early and late processing factors and partially processed pre-ribosomal RNAs (pre-rRNAs) leave the nucleolus at the same time.

Metaphase- the majority of processing components are associated with the surface of chromosomes as a perichromosomal region (PR).

Nucleolus in mitosisNucleolus in mitosis

Review: The multifunctional nucleolus www.nature.com/.../v8/n7/images/nrm2184-f6.jpg

Anaphase- cytoplasmic processing components become packaged in nucleolar-derived foci (NDF), whereas the other components remain around the condensed chromosomes. In late anaphase, cyclin B1–CDK1 levels decrease.

Early telophase- the number of NDF decreases and prenucleolar bodies (PNBs) are formed on the surface of each chromosome. The PR breaks down (indicated by an interrupted line) and processing components are taken up by PNBs. Nucleoli start to re-form around NORs of acrocentric chromosomes.

Late telophase- the nuclear envelope is re-formed and early (1) and late (2) processing components relocate in an ordered manner to the dense fibrillar component (DFC) and granular component (GC), respectively. CDK1, cyclin-dependent kinase-1; rRNA, ribosomal RNA.

The plurifunctional nucleolus

Major role as ribosome factories

Virus infection control: Many viruses target nucleolar functions Many viruses target nucleolar functions as part of their infectious strategy. The Rev protein of human as part of their infectious strategy. The Rev protein of human immunodeficiency virus (HIV) and the Rex protein of human T-immunodeficiency virus (HIV) and the Rex protein of human T-lymphotrophic virus (HTLV-I) depend on an interaction with B23, lymphotrophic virus (HTLV-I) depend on an interaction with B23, an endogenous cellular nucleolar protein .an endogenous cellular nucleolar protein .

Maturation of non-nucleolar RNAs or RNPs: e.g. RNase P RNA

Regulation of telomerase function

Tumor suppressor and oncogene activities

cell stress sensing and signaling

Role in cell senescence (aging)

Biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage

Involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs.

Regulation of cell cycle.

The Nucleolus and Human The Nucleolus and Human diseasesdiseases

The nucleolus has been linked to multiple forms of human The nucleolus has been linked to multiple forms of human disease.disease.

Multiple genetic disordersMultiple genetic disorders have been mapped to human genes have been mapped to human genes that encode nuclear proteins that are known to associate with that encode nuclear proteins that are known to associate with nucleoli under specific conditions, including nucleoli under specific conditions, including Werner syndrome Werner syndrome and Bloom syndromeand Bloom syndrome, more recently, , more recently, Rothmund–Thomson Rothmund–Thomson syndrome.syndrome.

Humoral hypercalcaemia of malignancyHumoral hypercalcaemia of malignancy is a common is a common complication of lung and certain other cancers. complication of lung and certain other cancers. PTHrPPTHrP was was first discovered as the first discovered as the hypercalcaemia factorhypercalcaemia factor that is produced that is produced by solid tumor. It localizes to the nucleus and the by solid tumor. It localizes to the nucleus and the nucleolus nucleolus in in certain tissues, such as skin, cartilage and bone. certain tissues, such as skin, cartilage and bone.

Diseases linked with aberrant ribosome biogenesis. Mutations in genes other than those that encode DNA

helicases also link the nucleolus to disease pathogenesis, as shown by Diamond–Blackfan anaemia.

ConclusionConclusion

► Nucleolus is a dynamic entity.Nucleolus is a dynamic entity.► Structure correlates with function.Structure correlates with function.► Has role in processes other than ribosome Has role in processes other than ribosome

biogenesis also.biogenesis also.► Still a lot to find out more about structure and Still a lot to find out more about structure and

function.function.► New imaging techniques will provide deeper New imaging techniques will provide deeper

understandingunderstanding

BibliographyBibliography Hernandez-Verdun, D. 2006a. [Hernandez-Verdun, D. 2006a. [http://www.springerlink.com/content/75n545v0g3186830http://www.springerlink.com/content/75n545v0g3186830 Nucleolus: From structure to Nucleolus: From structure to

dynamics. dynamics. Histochem Cell BiolHistochem Cell Biol 125: 127-137. Retrieved July 8, 2008. 125: 127-137. Retrieved July 8, 2008. Hernandez-Verdun, D. 2006b. Hernandez-Verdun, D. 2006b. The nucleolus: A model for the organization of nuclear functionsThe nucleolus: A model for the organization of nuclear functions. . Histochem Cell BiolHistochem Cell Biol

126: 135-148. Retrieved July 8, 2008. 126: 135-148. Retrieved July 8, 2008. Khadzhiolov, A. A. 1985. Khadzhiolov, A. A. 1985. The Nucleolus and Ribosome Biogenesis.The Nucleolus and Ribosome Biogenesis. Wien: Springer-Verlag. Wien: Springer-Verlag. ISBN 3211817905ISBN 3211817905. . Olson, M. O. J. 2004. Olson, M. O. J. 2004. The Nucleolus.The Nucleolus. Georgetown, TX: Landes Bioscience/ Eurekah.Com. New York: Kluwer Georgetown, TX: Landes Bioscience/ Eurekah.Com. New York: Kluwer

Academic/Plenum Publishers. Academic/Plenum Publishers. ISBN 0306478730ISBN 0306478730. . Olson, M. O. J., and M. Dundr. 2005. Olson, M. O. J., and M. Dundr. 2005. The moving parts of the nucleolusThe moving parts of the nucleolus. . Histochem Cell BiolHistochem Cell Biol 123: 203-216. Retrieved 123: 203-216. Retrieved

July 8, 2008.July 8, 2008.► McKeown PC, Shaw PJ.McKeown PC, Shaw PJ. ► Chromatin: linking structure and function in the nucleolus.Chromatin: linking structure and function in the nucleolus.► chromosoma, 2008 Oct 17 ,springerlink review.chromosoma, 2008 Oct 17 ,springerlink review.

► EMBO Rep. 2006 September; 7(9): 870–873. Published online 2006 August 18. doi: 10.1038/sj.embor.7400786.PMCID: PMC1559673Copyright © 2006, European Molecular Biology Organization

► Olson, M.O., M. Dundr, and A. Szebeni. "The Nucleolus: An Old Factory with Unexpected Capabilities." Trends in Cell Biology (2000) 10: 189-196.

► Websites► www.google.com► www.springerlink.com► www.ncbi.nlm.nih.gov