Nucleolus

The nucleolus is contained within the cell nucleus.

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 (also called nucleole) is a non-membrane bound structure found within the nucleus in which ribosomal RNA is transcribed, and is composed of protein and nucleic acids. The nucleolus ultrastructure can be visualized through an electron microscope while the organization and dynamics can be studied through fluorescent protein tagging and fluorescent recovery after photobleaching (FRAP). Malfunction of nucleoli can be the cause for several human diseases.

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. 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 and Hugh Wallace showed via hybridization experiments that nucleoli code for ribosomal DNA. Within the nucleus are one or more nucleoli. The nucleolus is roughly spherical, and appears as a mass of densely stained granules and fibres under an electron microscope. It consists of nucleolar organizers. They are specialized regions of some chromosomes with multiple copies of genes for ribosome synthesis, along with a considerable amount of RNA and proteins representing ribosomes in various stages of production. An average, healthy cell can produce up to 10 000 ribosomes per minute.

Function and ribosome assembly

Nucleoli are formed around specific genetic loci called Nucleolar Organizing Regions (NOR's), first described by Barbara McClintock. Because of this non-random organization, the nucleolus is defined as a 'genetically determined element' . A NOR is composed of tandem repeats of rRNA genes, which can be found in several different chromosomes. The human genome for example, contains more than 200 clustered copies of the rRNA genes 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, 5.8S, 28S) and an external non-transcribed spacer.

In the ribosome biogenesis, three eukaryotic RNA polymerases (pol I, II, III) are required which function in a coordinated manner. 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 RNA pol I. In humans, a similar PIC is assembled with SLI, the promoter selectivity factor (composed of TBP and TBP-associated factors, or TAFs), 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, 5.8S and 28S rRNA molecules. In eukaryotes, the RNA modifying enzymes are brought to their respective recognition sites through interaction with guide RNA's which bind these specific sequences. These guide RNA's belong to the class of small nucleolar RNA's (snoRNA's) which are complexed with proteins and exist as small-nucr-ribonucleoprotein (RNP) particles (snoRNP's). 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 localized 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 finds its way into the nucleolus to participate in the ribosome assembly. 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 '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.

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 nucleolar vacuole

A structure identified within the nucleolus 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 organization (FC, DFC, GC) of the nucleolus is commonly accepted, it has been proposed that this particular organization is only observed in higher eukaryotes and that it evolved from a bipartite organization with the transition from anamniotes to amniotes. Reflecting the substantial increase in the rDNA intergenic region, an original fibrillar component would have separated into the FC and the DFC.

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 rRNA genes may occasionally occur. This selective preference of transcription of rRNA genes is termed nucleolar dominance.