The nucleolus is contained within the cell nucleus. In Cell biology, the nucleus (pl nuclei; from Latin la ''nucleus'' or la ''nuculeus'' "little nut" or kernel is a membrane-enclosed

Schematic of typical animal cell, showing subcellular components. Organelles:
(1) nucleolus
(2) nucleus
(3) ribosomes (little dots)
(4) vesicle
(5) rough endoplasmic reticulum (ER)
(6) Golgi apparatus
(7) Cytoskeleton
(8) smooth endoplasmic reticulum (ER)
(9) mitochondria
(10) vacuole
(11) cytoplasm
(12) lysosome
(13) centrioles within centrosome

The nucleolus is a nuclear membraneless organelle in which the rDNA transcription takes place and in which the ribosomal subunits are assembled. In Cell biology, an organelle (pronunciation /ɔː(rgəˡnɛl/ is a specialized subunit within a cell that has a specific function and is usually separately enclosed In Cell biology, the nucleus (pl nuclei; from Latin la ''nucleus'' or la ''nuculeus'' "little nut" or kernel is a membrane-enclosed Ribosomes ( from ribo nucleic acid and "Greek soma ( meaning body") are complexes of RNA and Protein that A vesicle is a small bubble of liquid within a cell A more formal definition in Cell biology, would be that a vesicle is a relatively small intracellular membrane-enclosed The endoplasmic reticulum (Greek endo = "within" (prefix plásma = "formed entity" Latin reticulum = "little net" or ER, is an Organelle The Golgi apparatus (also called the cytoskeleton (also CSK is a cellular " Scaffolding " or " Skeleton " contained within the Cytoplasm. The endoplasmic reticulum (Greek endo = "within" (prefix plásma = "formed entity" Latin reticulum = "little net" or ER, is an Organelle In Cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed Organelle found in most eukaryotic cells. In general vacuole functions include Removing unwanted structural debris Isolating materials that might be harmful or a threat to the cell Containing The cytoplasm is the contents of a cell that is enclosed within the Plasma membrane. Lysosomes are Organelles that contain Digestive enzymes (acid Hydrolases. A Centriole is a barrel shaped Organelle found in most animal Eukaryotic cells though absent in Higher plants and Fungi. In Cell biology, the centrosome is an Organelle that serves as the main Microtubule organizing center (MTOC of the animal cell as well It is a highly dynamic structure from which the components are dispersed at the onset of mitosis and are reassembled at the end of cell division. Mitosis is the process in which a Eukaryotic cell separates the Chromosomes in its Cell nucleus, into two identical sets in two daughter nuclei The nucleolus can be visualised through electron microscopy while the organization and dynamics can be studied through Fluorescent Protein Tagging and Fluorescent Recovery after Photobleaching (FRAP). An electron microscope is a type of Microscope that uses Electrons to illuminate a specimen and create an enlarged image Malfunction of nucleoli can be the cause for several human diseases.

Introduction

The nucleolus (plural nucleoli) is a membraneless organelle of the cell nucleus, which in itself is an organelle. The main function of the nucleolus is the biogenesis and assembly of ribosome components (rRNA, ribosomal proteins). Ribosomes ( from ribo nucleic acid and "Greek soma ( meaning body") are complexes of RNA and Protein that Ribosomal RNA ( rRNA) is the central component of the Ribosome, the protein manufacturing machinery of all living cells. Ribosomes ( from ribo nucleic acid and "Greek soma ( meaning body") are complexes of RNA and Protein that During the initial stages of cell division, nucleoli are fragmented (they can no longer be seen in metaphase). Metaphase from the Ancient Greek μετά (after and φάσις (stage is a stage of Mitosis in the eukaryotic Cell cycle in which At the transition between telophase and interphase they reassemble around the chromatin regions where rDNA transcription is reinitiated. Telophase (sometimes spelled telephase) from the Ancient Greek "τελος" (end and "φασις" (stage is a stage in either Meiosis Interphase is the phase of the Cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for Cell The rDNA sequences encode the rRNA (ribosomal RNA) molecules of the ribosomes. Most plant and animal cells have one or more nucleoli, but some cell types do not have any. Since nucleoli carry out the production and maturation of ribosomes, large numbers of ribosomes are found inside them. In addition to ribosome biogenesis, nucleoli are believed to have other roles in cellular activity. Additionally, recent research pointed out that the nucleolus is also responsible for the trafficking of various prominent small RNA species. The nucleolus helps them during their maturation process and route to their final cellular destination. Moreover, although nucleoli become invisible during cell division, more recent studies have found that they are involved in cell cycle regulation. Several of its non-traditional roles include interaction with viral components, the regulation of tumor suppressor and oncogene activities, signal recognition particle assembly, the modification of small RNA strands, the control of aging and modulating telomerase function. An oncogene is a protein encoding Gene, which — when deregulated — participates in the onset and development of Cancer.

The nucleolus and rDNA transcription/rRNA processing/ribosome assembly

Nucleolus assembly occurs non-randomly. Nucleoli are formed around specific genetic loci called Nucleolar Organizing Regions (NOR’s). Because of this non-random organization, the nucleolus is defined as ‘a genetically determined element’ [6]. Previously described by Mc Clintock as the ‘Nucleolar Organizing Element’, a NOR is composed of tandem repeats of rRNA genes that are present in multiple copies throughout the genome. The human genome for example contains more than 200 copies of the rRNA gene and they are clustered on five different chromosomes. In a typical eukaryote, a rRNA gene consists of a promoter, internal and external transcribed spacers (ITS/ETS), rRNA coding sequences (18S, 5. In Biology, a promoter is a region of DNA that facilitates the transcription of a particular Gene. 8S, 28S) and an external ‘non’ transcribed spacer. ^[1]

In the ribosome biogenesis, three Eukaryotic RNA polymerases (pol I, II, III) are required which function in a coordinated manner. RNA polymerase ( RNAP or RNApol) is an Enzyme that produces RNA. In an initial stage, the rRNA genes are transcribed as a single unit within the nucleolus by RNA pol I. In order for this transcription to occur, several pol I-associated factors and rDNA-specific transacting factors are required. In yeast, the most important are UAF; upstream activating factor, TBP; tata-box binding protein and CF; core factor which bind promoter elements and form the pre-initiation complex (PIC), which is in turn recognized by pol I. Yeasts are a growth form of eukaryotic Microorganisms classified in the kingdom Fungi, with about 1500 Species currently described In humans, a similar PIC is assembled with SLI; the promoter selectivity factor which is composed of TBP and TBP-associated factors (TAF), IF; the transcription initiation factor and UBF; upstream binding factor.

Transcription of the ribosomal gene yields a long precursor molecule (45S pre-rRNA) which still contains the ITS and ETS. Further processing, which involves methylation and endo/exonuclease activity is therefore needed to generate the 18S rRNA, 5. Methylation is a term used in the chemical sciences to denote the attachment or substitution of a methyl group on various substrates. Exonucleases are enzymes (found as individual enzymes or as parts of larger enzyme complexes that cleave Nucleotides one at a time from an end of a polynucleotide chain 8S and 28S rRNA molecules. The RNA modifying enzymes are brought to their respective recognition sites through interaction with guide RNA’s which bind these specific sequences. The recognition sequence, also referred to as Recognition site, of any DNA-binding protein motif that exhibits binding specificity refers to the DNA sequence The guide RNA’s belong to the class of small nucleolar RNA’s (snoRNA’s) which are complexed with proteins and exist as small-nucleolar-ribonucleoprotein (RNP) particles (snoRNP’s). Small nucleolar RNAs (snoRNAs are a class of small RNA molecules that guide chemical modifications ( Methylation or pseudouridylation of Ribosomal RNAs Small nucleolar RNAs (snoRNAs are a class of small RNA molecules that guide chemical modifications ( Methylation or pseudouridylation of Ribosomal RNAs Once rRNA is processed, the rRNA molecules are ready to be assembled into ribosomes. However, an additional RNA molecule, the 5S rRNA, is necessary for this biogenesis. In yeast, the 5S rDNA sequence is localised in the external ‘non’ transcribed spacer and is transcribed in the nucleolus by RNA pol III. In higher eukaryotes and plants, the situation is more complex, for the 5S rDNA sequence lies outside the NOR and is transcribed in the nucleoplasm after which it is imported in the nucleolus to participate in the ribosome assembly. Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex Similar to the Cytoplasm of a cell, the nucleus contains nucleoplasm or nuclear sap. This assembly not only involves the rRNA, but ribosomal proteins as well. The genes encoding these r-proteins are transcribed by pol II in the nucleoplasm by a ‘conventional’ pathway of protein synthesis (transcription, pre-mRNA processing, nuclear export of mature mRNA and translation on cytoplasmic ribosomes). The mature r-proteins are then re-imported into the nucleolus. Association and maturation of rRNA’s and r-proteins result in the formation of the 40S and 60S subunits of the ribosome. These are exported through the nuclear pore complexes to the cytoplasm where they remain free or will become associated with the endoplasmic reticulum. The endoplasmic reticulum (Greek endo = "within" (prefix plásma = "formed entity" Latin reticulum = "little net" or ER, is an Organelle ^[1][2]

Morphology of the nucleolus

Nucleoli are typically composed out of three morphologically distinct regions which can be visualized by electron microscopy (EM) [3-7]:

1. An electron microscope is a type of Microscope that uses Electrons to illuminate a specimen and create an enlarged image Fibrillar Centers (FC):

• lightly stained when observed by EM
• composed of ‘fibrils’ (± 50Ǻ in Ø)
• presence of pol I and UBF
• multiple FC in one nucleolus
• accounts for only 1-2% of the total volume of the nucleolus

2. Dense Fibrillar Centers (DFC):

• surround the FC’s
• composed of ‘densely packed fibrils’ (30-50 Ǻ in Ø)
• occupies a large fraction of the nucleolus, ± 17% and roughly reflects the nucleolar engagement in ribosome biogenesis

3. Granular region (GR):

• region encompassing both the FC and the DFC
• consisting of granules 150-200 Ǻ in Ø
• granule rich region due to the presence of RNP particles
• with a fraction of about 75% it occupies the largest fraction of the total nucleolus volume
• although the nucleolus is not membrane-bound, due to the presence of GC the border with the surrounding chromatin and nucleoplasm is usually distinct. Chromatin is the complex basis of DNA and protein that makes up Chromosomes It is found inside the nuclei of eukaryotic cells, and within the

A substantial (additional) component of the nucleolus is chromatin which penetrates the organelle from the surrounding nucleoplasm. Chromatin is the complex basis of DNA and protein that makes up Chromosomes It is found inside the nuclei of eukaryotic cells, and within the

A continuous link between the nucleoplasm and the inner parts of the nucleolus exists through a network of nucleolar channels. In this way, macromolecules with a molecular weight up to 2000 kDa are easily distributed throughout the nucleolus. The unified atomic mass unit ( u) or Dalton ( Da) or sometimes universal mass unit, is an unit of Mass used to express

Remark: One last structure is identified within the nucleolus and is referred to as a nucleolar vacuole. There are multiple nucleolar vacuoles in the nucleolus, but it remains unclear whether they serve some functional/structural purpose or not. Although the ‘tripartite’ organisation (FC, DFC, GC) of the nucleolus is commonly accepted, it has been proposed that this particular organisation is only observed in higher eukaryotes and that it evolved from a bipartite organisation with the transition from anamniotes to amniotes. The amniotes are a group of Tetrapod Vertebrates that include the Synapsida ( Mammals and Mammal-like reptiles and Sauropsida Reflecting the substantial increase in the rDNA intergenic region, an original fibrillar component would have separated into the FC and the DFC^[7]. An Intergenic region is a stretch of DNA sequences located between clusters of Genes that contain few or no genes

Nucleolar organisation and dynamics

A mentioned previously, multiple nucleolar proteins and small nucleolar RNA’s (snoRNA’s) associate to form the processing machinery required in the ribosome biogenesis. They are involved in the modification of the nascent rRNA transcripts through methylation (2’-O-methylation/pseudouridylation) and endonucleolytic cleavage of the pre-RNAs. These processing stages are mainly confined in the DFC as revealed by the presence of these snoRNP (small-nuclear-ribonucleoprotein particles) constituting proteins, for example fibrillarin, nucleolin and U3 snoRNA. Fibrillarin, also known as FBL, is a human Gene. Nucleolin, also known as NCL, is a human Gene. Protein B23 and NOP52, involved in later stages of the processing are localized in the GC. This shows that the organization of the nucleoli is highly regulated and dependent on the stages in rRNA processing. These observations have also led to the hypothesis that rDNA transcription has to occur in the FC or at the junction between the FC and DFC because of the vectorial outward movement of the pre-RNA transcripts while they are processed to yield mature rRNAs.
If one considers the complete set of proteins and RNAs needed in the ribosome biogenesis, we can assume that a nucleolus is simply formed because certain proteins, involved in the transcription of the rDNA genes bind to their target regions, and that all around them there is a spontaneous assembly of all the elements involved in the modification of the nascent rRNA’s. Therefore, the organization occurs as a consequence of ribosome biogenesis. Several experimental approaches have been used to get a detailed view about this particular assembly process. The most important are: Fluorescent Protein Tagging, in which a protein of interest is fused with a fluorescent protein such as ‘green fluorescent protein’ (GFP) and Fluorescent Recovery After Photobleaching (FRAP) which consists of tagging a protein with a fusion protein after which the fluorescent molecules in the area of study are bleached with a laser. The green fluorescent protein ( GFP) is composed of 238 Amino acids (26 The fluorescent intensity of the area under study will recover because of outward diffusion of bleached molecules and inward diffusion of unbleached molecules. The former approach allows to keep track of the movement of the fluorescent complex (3D+time) and the latter allows measurement of the residence time (time spent in a certain area) of the fluorescent protein (ie. measuring the intracellular mobility).

Both methods rely on the ability of tagging a whole range of nucleolus associated proteins such as nucleolar proteins, histones, DNA binding proteins, transcription factors and spliceosomes. In Biology, histones are the chief Protein components of Chromatin. In the field of Molecular biology, a transcription factor (sometimes called a sequence-specific DNA binding factor is a Protein that binds to specific sequences A spliceosome is a complex of specialized RNA and Protein subunits that removes Introns from a transcribed pre- mRNA ( HnRNA Tracking and measuring the residence time of the tagged proteins allowed demonstration of the rapid association/dissociation rates of nucleolar proteins with other nucleolar components, the continuous exchange of proteins between the nucleolus and the nucleoplasm during interphase and the involvement of these nucleolar proteins with other nuclear domains. It has for example been found that Cajal Bodies (CB) are enriched in small nuclear and nucleolar ribonucleoprotein and that they contain several nucleolar associated processing proteins such as fibrillarin. Cajal bodies (CBs are spherical sub-organelles found in the nucleus of proliferative cells like Tumor cells or metabolically active cells like Neurons Therefore it has been proposed that there should exist a functional relationship between nucleoli and Cajal Bodies. ^[3,4]

Several experimental observations indicate that the recruitment of the nucleolus constituting elements occurs non-random and that it is regulated by the cell cycle progression. During mitosis, the transcription machinery stays closely associated with the rDNA. Mitosis is the process in which a Eukaryotic cell separates the Chromosomes in its Cell nucleus, into two identical sets in two daughter nuclei However, the transcription is repressed by the cyclin B/Cdk1 protein kinase complex (PMF). Cyclin B is a member of the Cyclin family Cyclin B is a mitotic cyclin Cell division cycle 2 G1 to S and G2 to M, also known as Cdk1 (CDC2, is a human Gene. This complex is activated at the onset of mitosis and represses nuclear activities by phosphorylating a number of protein kinases or structural proteins involved in the cellular rearrangements needed for proper cell division. It is at the end of Mitosis, when the PMF is degraded through proteolytic cleavage of cyclin B, that nucleoli reassemble around the rDNA sites in a response to the reinitiating of rDNA transcription. A protease is any Enzyme that conducts Proteolysis, that is begins protein Catabolism by Hydrolysis of the Peptide bonds that link The nucleolar proteins are, in contrast to the proteins involved in the transcription, localized at the periphery of the chromosomes during the M phase of the cell cycle. Mitosis is the process in which a Eukaryotic cell separates the Chromosomes in its Cell nucleus, into two identical sets in two daughter nuclei This can be visualized by Fluorescent Protein Tagging. At the transition of telophase to G1, the majority of them are grouped into Prenucleolar Bodies (PNB). It is these PNB that perform the translocation from the chromosomes to the sites were rDNA transcription has started. The PNB’s are thought to function as an assembly platform and as a reservoir for protein complexes which release the processing proteins at the sites of rDNA transcription. Early processing proteins such as fibrillarin are recruited in a response to a decrease in cyclin B/Cdk1 activity whereas late processing proteins such as B23 and Nop52 are recruited in a response to cyclin dependent kinase (cdk) activity. Cyclins are a family of Proteins involved in the progression of cells through the Cell cycle. In this way, the various processing protein can be released exactly at the time they are needed during rRNA synthesis. ^{[3, 4]}

Human diseases associated with the nucleolus

Human diseases associated with a malfunction of the nucleolus can be caused by viral infections, increased nucleolar activity or simply by congenital mutations affecting the nucleolar proteins.

• If a virus contains a NOS (nucleolar targeting signal) in its genome, some viral particle will be directed towards the nucleolus. Such is the case with the Human Immunodeficiency Virus (HIV) which directs the HIV-1 Rev protein to the nucleolus. Human immunodeficiency virus ( HIV) is a Lentivirus (a member of the Retrovirus family that can lead to acquired immunodeficiency syndrome Through interaction with the B23 nucleolar protein it serves its purpose by regulating the splicing pattern of the HIV-1 mRNA, for it promotes the export of unspliced mRNA to the cytoplasm. The cytoplasm is the contents of a cell that is enclosed within the Plasma membrane. It has been proposed that the Rev protein is localised in the nucleolus to provide an alternative translocation pathway for viral (unspliced/partially spliced) mRNA from the nucleoplasm to the cytoplasm. In this way the viral mRNA is protected against degradation (which would normally take place to protect the cell against the translation of pre(unprocessed)-mRNA).

• An increased nucleolar activity will have its effect on the overproduction of ribosomes which eventually will lead to tumorgenesis and cancer. A key factor in these dysfunctional nucleoli is the protein c-myc, product of the c-myc-proto-oncogene. It stimulates the ribosome biogenesis by directly regulation pol I, influencing the transcription of pol II, III and by associating with the SL1 component of the pre-initiation complex which increases the efficiency of the recruitment of pol I to the pre-initiation complex.
  
  
• In addition, several congenital mutations affecting the nucleolar proteins have been described; Weine syndrome, Treacher Collins syndrome and dyskeratosis congenital syndrome. ^{[3, 4, 6]}

Nucleolar dominance

Nucleolar dominance has also been shown for rRNA genes. In some organisms, particularly plants, when two nuclei are combined into a single cell during hybridization the developing organism can 'choose' one set of rRNA genes for transcription. The rRNA genes of the other parent are suppressed and not generally transcribed, though reactivation of the suppressed or 'inferior' rRNA genes may occasionally occur. This selective preference of transcription of rRNA genes is termed nucleolar dominance.

History

John Gurdon and Donald Brown generated the first interest in cell nucleoli in 1964, when they discovered them in the African clawed frog Xenopus laevis. Sir John Bertrand Gurdon, FRS (b 2 October 1933) is a British developmental biologist. Donald E Brown is an American professor of Anthropology ( Emeritus) The African clawed frog ( Xenopus laevis, also known as platanna) is a species of South African aquatic Frog of the genus Xenopus They found that 25% of the frog eggs had no nucleolus and that such eggs were not capable of life. Half of the eggs had one nucleolus and 25% had two. They concluded that the nucleolus had a function necessary for life. In 1966 Max L. Birnstiel und Hugh Wallace showed via hybridization experiments that nucleoli code for ribosomal DNA.

See also

• Transcription (genetics)
• Cancer
• RNA
• Paraspeckle

References

3. Hernandez-Verdun, D. (2006) Nucleolus: from structure to dynamics . Histochem Cell Biol 125: 127-137 [1]
4. Hernandez-Verdun, D. (2006) The nucleolus: a model for the organisation of nuclear functions. Histochem Cell Biol 126: 135-148 [2]
5. Olson, M. O. J. , Dundr, M. , (2005) The moving parts of the Nucleolus. Histochem Cell Biol 123: 203-216 [3]
6. Raška, I. Shaw, P. J. , Cmarko, D. (2006) New Insights into Nucleolar Architecture and Activity [4]
7. Thiry, M. , Lafontaine, L. J. (2005) Birth of a nucleolus: the evolution of nucleolar compartments. TRENDS in Cell Biology 15 [5]
8. Olson, Mark O. J. (2004). The Nucleolus Georgetown, Texas : Landes Bioscience / Eurekah. Com. Kluwer Academic/Plenum Publishers. New York. ISBN 0-306-47873-0
9. Khadzhiolov, Asen A. (1985). The nucleolus and ribosome biogenesis Wien : Springer-Verlag. ISBN 3-211-81790-5
10. Thiry, Marc & Guy Goessens (1996). The nucleolus during the cell cycle Hong Kong : Springer ; Austin, Tex.  : R. G. Landes Company. New York. ISBN 3-540-61352-8

External links

• Nucleolus under electron microscope II at uni-mainz.de
• Recent concerns about nucleolus at hgu.mrc.ac.uk
• MeSH Cell+Nucleolus
• Histology at BU 20104loa