Nucleolus: Ultra Structure

Ultra Structure of Nucleolus

The nucleolus is a tri-molecular nuclear body that participates in rRNA synthesis and maturation, ribosome subunit assembly and export, cell cycle regulation, stress sensing, and response. The nucleolus consists in general terms of transcriptionally active and inactive compartments, which have been termed Fibrillar Centers and Dense Fibrillar Components, the Granular Component is primarily a packaging area and the Nucleolar Matrix is a supporting structure. The nucleolus of different eukaryotes varies greatly in complexity and structure but the major function is conserved and all nucleoli possess 30-60 tandem arrays of rDNA genes which get activated during nucleolus formation. The typical effect of inhibition of RNA polymerases that transcribe rRNA is disassembly of the nucleoli. The regions of nucleolar rDNA genes that get transcribed are seen as amorphous Fibrillar Centers surrounded by the Dense Fibrillar Components where nascent transcripts are modified, folded and assembled together with RNA-binding proteins and some small nucleolar RNPs.

Composition of the Nucleolus:

The nucleolus is a unique organelle, composed of diverse macromolecular components and hosted in the nuclear environment. The nucleolus is a highly complex structure whose components vary not only in nature and abundance but also in distribution and characteristics. Ultrastructural studies definitively showed that the nucleolus is a nuclear subcompartment formed by specific combinations of nucleolar proteins and RNAs. However, it is also the consequence of a particular activity in the synthesis and processing of pre-ribosomal RNAs.Of these components, the RNA molecules are the most abundant. However, it is also the consequence of a particular activity in the synthesis and processing of pre-ribosomal RNAs. Biochemical studies have identified many proteins in nucleolar fractions, some of which are either ribosome subunit components or assist the assembly of ribosomes and are needed for rRNA synthesis and processing. Large-scale analyses and bioinformatics have identified further nucleolar proteins that either localize to the nucleolus in steady-state conditions or localize after specific stimuli. In addition to certain proteins associated with a subtype of phosphoribosyl pyrophosphate amidotransferase, there are other enzymes relatively strictly related to nucleolar functions. For example, some enzymes associated with cellular metabolism participate in rRNA synthesis. GTPases act in other pathways related to ribosome biogenesis.Another set of proteins associated with ribosome biogenesis are zinc finger proteins because they are essential for proper protein synthesis due to their direct involvement in ribosome integrity. A large number of RNA helicases involved in ribosome synthesis interact with two types of rRNA: one is associated with a specific snoRNA, and the other is associated with the 18S rRNA. This suggests that their activities are necessary at both sites and, therefore, during nucleolar foci formation and swells. Among the highly conserved proteins, there are also some Argonautes, which are ribonucleoproteins implicated in the silencing of transposable elements by interacting with specific RNA molecules. Along with the RNA helicases just mentioned, certain enzymes also interact with rRNA.

Nucleolar Structure: The nucleolus is a spherical non-membrane-bounded structure that is present in the nuclei of eukaryotes. However, among prokaryotes, it has been reported to be absent. Its structure usually appears formed by a filamentous substance. This structure is present regardless of the activity of the cell cycle. As eukaryotes carry out a protein synthesis process through the ribosome, they evolve and develop their cellular composition along with the respective increase in RNA, given that the nucleolus is where the synthesis of the ribonucleoproteins that evolve and form the components of ribosomes takes place.

The nucleolus is a dynamic structure that varies in size throughout the cell cycle due primarily to differences in the synthesis and processing of ribosomal RNA. It is also accepted that some of the RNA-binding proteins required for ribosome biogenesis and assembly arise at the nucleolus, but it is important to note that some of these also traffic out of the nucleus into the nucleoplasm and cytosol. These proteins additionally carry signals for the subsequent import into the nucleolus at the appropriate stage. The nucleolus is broadly divided into substructures: the fibrillar center, the dense fibrillar component, and the granular component. The fibrillar center is a low-DNA content, and the nucleoli of all vertebrates contain a cluster of rRNA genes organized in tandem on a chromosome. It is the sites of active rRNA transcription by RNA polymerase I, and the other two are involved in rRNA processing and post-transcriptional modifications, pre-rRNA hypermethylation, and pseudouridylation. The dense fibrillar component contains the nascent and processing ribosomal RNA transcript and is rich in snoRNPs that modify and process pre-rRNA, whereas the granular component contains pre-60S and pre-40S ribosomal particles that exit from the nucleolus and mature in the cytoplasm.

Fibrillar Center Fibrillar centers, the sites of ribosomal gene organization into transcriptionally inactive, non-nucleosomal chromatin loops hosting ribosomal RNA gene specific polymerases, implicated in pre-rRNA transcript synthesis, had been visualized in many vertebrates through immunogold EM cytochemistry of specific antibody against the RNA polymerase I largest subunit. This visualization executed in small nucleoli and from the different tissue types of adults had demonstrated eminent invariability of fibrillar center number, as well as the fact that there are enough polymerases available during cell cycle and upon the mitosis to fit with possible massive nucleolar transcription. This ribosomal RNA gene loci polymerase association had been found also in frequently observed and apparently rhythmically activated yeast nucleolar structures of unfibrillated cords and lampbrush chromosomes, looped out of nucleosomal chromatin. Recent cryo-electron tomography data confirmed that the intermediates of highly polymerized pre-rRNA transcript synthesis are really shaped like unclosed, Uarin-like, RNA-protein decamers. Fibrillar center structure damage indirectly effects surrounding dense fibrillar and granular components, which in their turn appear to influence the ribosome biosynthesis velocity. Based on all these elements, we suggest the proposal that the fibrillar center seems to be a molecule of some kind of scaffolded chromatin, holding polymerized pre-rRNA decameric chains in places longest in the cell where pre-rRNA transcriptions, but not the cleavage of the pre-rRNA and/or ribosome assembly occurs.

Dense Fibrillar Component Dense fibrillary components are fibrillar masses lying in a space between amorphous fibrillar centers and granular components. The term "dense fibrillar component" is exclusively used in the context of nucleolar ultrastructure; the general term “dense fibrillar region”, however, is also used. In C. elegans, rats and mice, the dense fibrillar components were first identified as fibrillar electron-dense masses resolvable from amorphous fibrillar centers and granular components in nuclear sections. The term was used to describe the electron-dense particulate bodies observed in the transpired electron micrographs of purified nucleolar fractions inducing same amounts of RNA synthesis in reticulocytes as whole nucleoli, but reducing to 50% the cytoplasmic levels of 28S rRNA. The observation that these dense bodies became less dense during the incubation of reticulocyte nuclei with an exogenous RNA polymerase I transcription system and increased their density during the subsequent RNA synthesis, prompted the formulation of a model of formation and accumulation of nascent rRNA in the dense fibrillar components. The fibrillar bodies became less dense again during the aging and cessation of rRNA synthesis in the nucleolus, causing a decreased efficiency of RNA synthesis in reticulocyte nuclei.

The main molecular constituents of the dense fibrillar components are found within the fibrillar bodies visualized in autoradiographs of reticulocyte nucleolar sections treated with 3 H-uridine and exposed to nuclear polyadenylated RNA abundance. The majority of the abundant RNA molecules are ready to be processed into rRNA and the ribosome system. The ribonucleoproteins found in the fibrillar bodies of the dense fibrillar components visualized in the tied RNA/Nops immunocytochemical localization or affinity techniques, and in mass spectrometric analyses of isolated nucleolar fractions, are in agreement with the two distinct RNA pairs previously found. One pair of RNA -yctokeratins are associated with Scotch-taped and tulip nucleolar forms throughout the rRNA cycle, but are depleted from scalloped nucleolar forms semi-persisted during the transcription inhibition-induced RNA cycle or induced during de novo nucleolar formation. These predictions ought to be experimentally ratified in the future.

Granular Component Granular component is the last and also the most complex stage of nucleolar organization. Granules from GP presented with heterogeneous sizes, shapes, and in different aggregation states. Granular component granules were formed usually with a dense center and a clear halo on the circumference, in addition to the ribosomal subunits and premade rRNA precursors, regional GC also had non nucleolar items like heterochromatin patches, probably to promote rRNA elements exit around nucleolus to nurse onmogenesis filament. The lack of RNase areas within GP indicated that granules newly synthesized monomers waiting configured nucleolar bodies. Recent cryogenic electron tomography of electron-dense particles of GP obtained from transformation cells as well as some GP particle structures obtained from filamentous cells during activation of cell proliferation revealed that GP is a specific pre-ribosomal subunit assembly center. The pre-ribosomal subunits including 90S with rind-like particles whose fundamental structural unit is the protein molecule, are disposed of labyrinthine pattern display on ring-shaped particle core. These GP particles are nucleus-specific organelles which generate near 70S ribosome subunits in the nucleoplasm of some species cells.

Granular component is generated and assembled progressively in definitive process. The two processes of GP nuclear body formation and ribosome assembly converge at the spherical ribonucleoprotein particles, or pre-ribosomes. The evidence indicates that the particles containing preribosomal RNA are the final stages of preribosome construction. The granular component would be several compositions or more which could be assembling and disassembling guided by prerRNA elements other than preribosomes and ribosome subunits substructures aspectsponsor roles of the particles provided by prerRNA elements for the substratum. Granular component is formed by the preribosomal bigger spheres or specific bodies of nucleolus.