The Nucleus

A very brief introduction to nucleus structure and function.

1. The nucleus Presented By: Group 1 Afsha Zia, Alizay, Ayesha Khalid, Noor-ul-naeem, Tehreem Ishtiaq, Tehreem Tariq

2. Contents Introduction Nuclear Envelope Transport T hrough Nucleus Internal Organization of Nucleus Assembly of RNAs

3. The Nucleus When you look at a eukaryotic cell in a light microscope nucleus is the largest visible compartment. The presence of a nucleus distinguishes eukaryotic cells from prokaryotic cells. The nucleus houses all of the eukaryotic cell’s genome and acts as a center for controlling cellular activities. Processes such as DNA replication, transcription, and RNA processing all takes place within the nucleus.

4. Discovery Antony van Leeuwenhoek ( 1632–1723) was probably the first to observe nucleus in the blood cells of birds and amphibians. But Felice Fontana ( 1730–1805) was the actual discoverer of nucleus by observing epidermal cells of eel. The Scottish botanist, Robert Brown ( 1773–1858) observed the nucleus in plant cells and was the first to call these structures ‘nuclei’.

5. Structure A double membrane called nuclear envelope encloses the nucleus. The lumen separates the two membranes and is continuous with the Endoplasmic Reticulum. Macromolecules pass between the nucleus and cytoplasm through the Nuclear P ore complexes (NPCs) that are channels spanning the envelope. The nucleus has non-enveloped sub-compartments with specialized function. Nucleolus is the most clearly visible structure. Regions other than the nucleolus are referred to as the nucleoplasm . Other sub-compartments include speckles, cajal bodies and PML bodies etc. Inside the nucleus the DNA can be found in the compacted and highly stained form , heterochromatin or in the less densely compacted form the euchromatin .

6. Structure

7. Advantages to eukaryotes in having a nucleus By separating the genome from the cytoplasm, the nuclear envelope allows gene expression to be regulated by mechanisms that are unique to eukaryotes. In prokaryotes mRNAs are translated before their transcription is completed, eukaryotic mRNAs are produced from precursor RNAs through complete processes before being transported from the nucleus to the cytoplasm. When DNA is replicating during interphase the nucleus protects the relaxed DNA from breakage by force of cytoskeleton .

8. Nuclei Vary In Number and Appearance Nuclei can vary in size according to the amount of DNA they contain. The single celled organism Saccharomyces cerevisiae has a nucleus of 1 µ in diameter. Many multicellular organisms have a nucleus of size 5-10µ in diameter . The percentage of volume occupied by nuclei in different cells varies. Volume occupied by nucleus in yeasts cells is 1%-2%, 10% in somatic cells and 40%-60% in cells that do not have many cytoplasmic functions .

9. In most cells the nucleus is oblong or oval shaped to minimize surface area of enclosure. We can identify different cells from the shape of their nucleus. Example : During leukemia an excess of white blood cells are produced which have different shapes of nuclei at different stages of development. The type of leukemia can be diagnosed by studying the morphology of nuclei of the cells produced in excess . Most cells are mononucleate , some are multi nucleate and some are anucleate . Example : Multinucleate cells include those of Drosophila melanogaster in embryonic stages. Myocytes formed by fusion of myoblasts are also multinucleate. Cells like the mammalian red blood cells and cells of lens of vertebrate eye lack nucleus . Figure : A Drosophila embryo at the multinucleate stage

10. Nuclear envelope

11. Nuclear membrane Nuclear Membrane is a fence between nucleus and cytoplasm to stave off free transmission of molecules. It provides nucleus an identity of separate biochemical compounds. The nuclear membrane consists of: Outer nuclear membrane I nner nuclear membrane Perinuclear space Nuclear pores Nuclear lamina

12. Outer nuclear membrane The outer nuclear membrane is continuous with endoplasmic reticulum, therefore the lumen of nuclear membrane is directly connected with lumen of ER. The outer nuclear membrane is functionally homologous to ER membrane. The cytoplasmic surface of outer nuclear membrane has ribosomes that are different in composition of protein and these ribosomes are enriched in membrane proteins (for cytoskeleton binding).

13. Perinuclear Space Space is present between ONM and INM and is called Perinuclear space or lumen of envelope . The thickness of each nuclear membrane is 7-8nm thick while perinuclear space is 20-40nm thick .

14. Inner nuclear membrane Proteins that are specific to nucleus are present in INM such as those that bind the nuclear lamina. Including Lamin B receptor (LBR), lamina-associated polypeptide (LAP) 1, LAP2, emerin, MAN1 and nurim. Most of these proteins interact with lamins and chromatin. Mutations in emerin and nuclear lamins have been associated with muscular dystrophies and lipodystrophy . Integral proteins of the inner nuclear membrane are synthesized on the rough ER and reach the inner nuclear membrane by lateral diffusion in the connected ER and nuclear envelope membranes.

15. Nuclear pore channel (NPC ) The Phospholipid bilayer is only permeable for non-polar micromolecules. The only channel through which transmission of polar micromolecules and macromolecules occurs is through Nuclear Pore Complex. NPCS are the points where lNM and ONM are continuous . Figure: The inner and outer membranes of the nuclear envelope are fused at the NPC.

16. Nuclear Lamina I n multicellular eukaryotes, a fibrous mesh work supports the inner nuclear membrane called Nuclear Lamina The nuclear lamina is present inside the nuclear envelope. Lamins are 60-80 kilo Dalton fibrous proteins that makeup the nuclear lamina Some associated proteins are also present. Lamins belong to a class of intermediate filament proteins. Nuclear Lamina disease: Emery-Dreifuss muscular dystrophy Hutchinson-Gilford progeria syndrome

17. Model of Lamin Assembly Association of lamins with each other occurs to form higher order structure called nuclear lamina. Attachment of lamins to the nuclear envelope is mediated by prenylation and binding to specific inner nuclear membrane proteins such as emerin and Lamin B receptor .

18. During Mitosis the Nuclear Envelope Disassembles Onset The nuclear membrane disassembles and the nuclear lamina depolymerizes due to phosphorylation of nuclear lamins by C yclin-dependant protein kinase . The nuclear envelope membrane proteins also dissemble by phosphorylation leading to disassembling of NPCs. NPC proteins become bound to nuclear import receptors, which play an important part in the reassembly of NPCs at the end of mitosis. Motor proteins on microtubules also help in tearing down the nuclear membrane. Offset The nuclear envelope forms around chromatin. Chromatin is surrounded by a shroud of Ran-GTP. The assembly of NPCs is started by nuclear import receptors from NPC proteins displaced by the Ran-GTP cloud. Alongside the nuclear envelope membrane proteins with dephosphorylated lamins start attaching to chromatin again. Membranes from ER fuse over the chromatin until a complete Nuclear envelope is formed.

19. Nuclear Pore: transport through nucleus

20. What are Nuclear Pores? Small polar molecules, ions and macro-molecules can only move in between the nucleus and cytoplasm through channels. The large circles with diameter of 120nm and molecular mass of ̴125 million Dalton and 30X the size of ribosomes are pores that are collectively called as Nuclear Pore complex . They are composed of several proteins of 30 different types. Those specialized proteins are named as Nucleoporins .

21. variation in number of NPCs in membranes Requirement of nuclear transmission of a cell decides the number of NPCs. 150-250 NPCs are present in the much smaller yeast cell's nuclear envelope. Some cells contain millions of NPCs (e.g. Xenopus ) because they are very active transcriptionally and translationally . FIGURE The surface of the nuclear envelope of a Xenopus laevis oocyte is covered with NPCs.

22. Model of the Nuclear pore complex The complex consists of an assembly of eight spokes attached to rings on the cytoplasmic and nuclear sides of the nuclear envelope. The spoke-ring assembly surrounds a central channel containing the central transporter . Cytoplasmic filaments extend from the cytoplasmic ring, and filaments forming the nuclear basket extend from the nuclear ring .

23. Functions of Nuclear pore Nuclear pores play an important role in physiology of eukaryotic cells by controlling the traffic of molecules between nucleus and cytoplasm. RNAs that are synthesized in nucleus are carried out through the nuclear pores in order to synthesize proteins in the cytoplasm. Conversely, proteins required for nuclear functions (e.g., transcription factors) must be transported to the nucleus from their sites of synthesis in the cytoplasm. Many proteins shuttle continuously in between nucleus and cytoplasm which is also a very specialized function of nuclear pore.

24. Molecular traffic through nuclear pore complexes Small molecules are able to pass rapidly through open channels in the nuclear pore complex by  passive diffusion . In contrast, macromolecules are transported by a selective, energy-dependent mechanism that acts predominantly to import proteins to the nucleus and export RNAs to the cytoplasm.

25. Nuclear localization signals Figure: The nuclear localization signal of nucleoplasm is bipartite, consisting of a Lys-Arg sequence, followed by a Lys-Lys-Lys-Lys sequence located ten amino acids farther downstream . These proteins are targeted to the nucleus by specific amino acid sequences called nuclear localization signals. These localization signals are detected by nuclear transport receptors (Importins) as they carry proteins into the nucleus. They direct protein transport through nuclear pore complex. Nuclear localization signals have yet been observed in many proteins, and amino acids that are involved in these localization signals are found close to each other but not immediately adjacent to each other.

26. Transport of RNA s Since proteins are synthesized in the cytoplasm, the export of mRNAs, rRNAs, tRNAs, and microRNAs (miRNAs) is a critical step in gene expression in eukaryotic cells. As proteins are selectively transported from cytoplasm to nucleus in the same way RNAs are transported from nucleus to cytoplasm to synthesize these proteins. Like protein import, the export of all RNAs through the nuclear pore complex is an active, energy-dependent process requiring the transport receptors to interact with the nuclear pore complex. Karyopherin, importins and exportins transport most tRNAs, rRNAs, miRNAs and small nuclear RNAs. In contrast to mRNAs, tRNAs, and rRNAs, which function in the cytoplasm, many small RNAs (snRNAs and snoRNAs) function within the nucleus as components of the RNA processing machinery.

27. Transport of s n RNA s between nucleus and cytoplasm snRNAs firstly get transported into the cytoplasm from nucleus. Then they associate with proteins to form functional snRNPs . And then go back to the nucleus. Crm1 and other transport receptor proteins that bind to the 5' 7-methylguanosine caps of snRNAs are involved in the export of the snRNAs to the cytoplasm . Figure: Small nuclear RNAs are initially exported from the nucleus to the cytoplasm, where they associate with proteins to form snRNPs. The assembled snRNPs are then transported back into the nucleus.

28. Active Transport of macromolecules Uncharged molecules including water, can diffuse freely through phospholipid bilayers. Other macromolecules that are transported across the nuclear envelope move through NPCs. The process of moving through the NPC is called translocation .

29. Internal organization of nucleus

30. Internal organization of nucleus A loosely organized matrix of nuclear lamins extends from nuclear lamina into the interior of nucleus in animal cell, which serves as sites of chromatin attachment and bind other proteins into the nuclear bodies . Chromatin is organized into large loops of DNA and regions of these l oops are bound to the lamin matrix by lamin binding proteins . Many other nuclear proteins form Lamin-dependent complex

31. Chromosomes And Higher Order Chromatin Structures During interphase the heterochromatin remains highly condensed and inactive and euchromatin is de-condensed and distributed throughout the nucleus, a heterochromatin is of two types: Constitutive heterochromatin remains permanently in heterochromatin stage, having DNA sequences that are not transcribed. Facultative heterochromatin consist of euchromatin that takes on the staining and compactness characteristics of heterochromatin during some phase of development, having DNA sequences that are transcribed in cell which we are studying but are transcribed in other cells .

32. Although interphase chromatin appears to be uniformly distributed, the chromosomes are actually arranged in an organized fashion and divided into discrete functional domains that play an important role in regulating gene expression. Heterochromatin in interphase nuclei The euchromatin is distributed throughout the nucleus. The heterochromatin is indicated by arrowheads and the nucleolus by an arrow .

33. Sub-compartments within the nucleus The internal organization of nucleus is the result of localization of nuclear processes to specific regions of nucleus . Many enzymes and proteins of nucleus are organized to discrete sub-nuclear bodies . The nature and function of these nuclear bodies are not clear. Replication of multiple DNA molecules takes place on the cluster site of nuclei in mammalian cell. Sub-compartments within the nucleolus, as visualized by electron microscopy.

34. Actively transcribed genes appears to be distributed throughout the nucleus. Components of mRNA splicing machinery are concerted in nuclear speckles . Immunoflourescent staining showed that rather than being distributed uniformly throughout the nucleus the components of RNA splicing apparatus are concerted in these 20-50 discrete structures: Speckles : storage sites of splicing components where pre mRNA processing occurs. In addition to speckles nuclei also contain PML and cajal bodies. PML bodies : transcription factors & chromatin-modifying enzymes localize here. Cajal bodies/ coiled body : involved in snRNP biogenesis, histone mRNA processing & telomere maintenance. Gemini Bodies : are not found in all cells, and some of their components are also found in Cajal bodies, suggesting they may not perform distinct functions. Cajal bodies and Gemini bodies can be detected by using specific antibodies and indirect immuno - fluorescence .

35. Chromosomes occupy distinct territories Although the nucleus lacks internal membranes, nuclei are highly organized and contain many sub-compartments . Each chromosome occupies a distinct region or territory , which prevents chromosomes from becoming entangled with one another. The nucleus contains both chromosome domains and inter-chromosomal regions. Individual chromosomes occupy distinct areas of the nucleus called chromosome territories.

36. N uclear sub compartments are not membrane-bounded Nuclear sub compartments are not membrane-bounded. rRNA is synthesized and ribosomal subunits are assembled in the nucleolus. Genes that encode runs are present on multiple chromosomes that cluster together to form nucleoli sub-compartments , mRNA splicing factors are stored in nuclear speckles and move to sites of transcription where they function. Other nuclear bodies have been identified using antibodies; some of these bodies are believed to concentrate specific nuclear proteins, but the functions of most nuclear bodies are unknown.

37. nucleolus

38. The Nucleolus The most prominent nuclear body is the nucleolus . It is the site of rRNA transcription and processing as well as aspects of ribosome assembly . The nucleolus is a ribosome production factory, designed to fulfill the need for regulated and efficient production of rRNAs and assembly of the ribosomal subunits . Actively growing mammalian cells, for example, contain 5 million to 10 million ribosomes that must be synthesized each time the cell divides. Recent evidence suggests that nucleoli also have a more general role in RNA modification and that several types of RNA move in and out of the nucleolus at specific stages during their processing. Nucleoli in amphibian oocytes The amplified rRNA genes of Xenopu s oocytes are clustered in multiple nucleoli (darkly stained spots).

39. Ribosomal RNA Genes and the Organization of the Nucleolus The nucleolus is associated with the chromosomal regions that contain the genes for the 5.8S, 18S, and 28S r RNAs. Ribosomes of higher eukaryotes contain four types of RNA designated the 5S, 5.8S, 18S, and 28S rRNAs . The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit within the nucleolus by RNA polymerase I, yielding a 45S ribosomal precursor RNA. Transcription of the 5S rRNA, which is also found in the 60S ribosomal subunit, takes place outside the nucleolus in higher eukaryotes and is catalyzed by RNA polymerase III .

40. rRNA Genes The human genome, for example, contains about 200 copies of the gene that encodes the 5.8S, 18S, and 28S rRNAs and approximately 2000 copies of the gene that encodes 5S RNA. The genes for 5.8S, 18S, and 28S rRNAs are clustered in tandem arrays on five different human chromosomes (chromosomes 13, 14, 15, 21, and 22) The 5S rRNA genes are present in a single tandem array on chromosome 1.

41. Ribosomal RNA genes Each rRNA gene is a single transcription unit containing the 185, 5.85, and 285 rRNAs as well as transcribed spacer sequences . The rRNA genes are organized in tandem arrays , separated by non-transcribed spacer DNA

42. Importance of ribosome production The importance of ribosome production is particularly evident in oocytes in which the rRNA genes are amplified to support the synthesis of the large numbers of ribosomes required for early embryonic development . In Xenopus oocytes, the rRNA genes are amplified approximately two-thousand-fold, resulting in about one million copies per cell . These amplified rRNA genes are distributed to thousands of nucleoli, which support the accumulation of nearly 1012 ribosomes per oocyte . Recently,  it has been shown that ribosome biogenesis is intimately linked to multiple cellular signaling pathways and that defects in ribosome production can lead to a wide variety of human diseases.

43. Transcription and Processing of rRNA Each nucleolar organizing region contains a cluster of tandemly repeated rRNA genes separated from each other by non-transcribed spacer DNA. These genes are very actively transcribed by RNA polymerase I, allowing their transcription to be readily visualized by electron microscopy. In such electron micrographs, each of the tandemly arrayed rRNA genes is surrounded by densely packed growing RNA chains forming a structure that looks like a Christmas tree. The high density of growing RNA chains reflects that of RNA polymerase molecules, which are present at a maximal density of approximately one polymerase per hundred base pairs of template DNA .

44. Ribosome assembly Early in ribosome assembly, the processing of the two nascent ribosomal subunits occur separately Processing of the smaller subunit , which contains only the 18S rRNA, is simpler and involves only four endonuclease cleavages . In higher eukaryotes, this is completed within the nucleus but in yeast the final cleavage to the mature 18S rRNA actually occurs after export of the 40S subunit to the cytosol . Processing of the larger subunit , which contains the 28S, 5.8and 5S rRNAs, involves extensive nuclease cleavages and is completed within the nucleolus. Consequently, most of the pre-ribosomal particles in the nucleolus represent precursors to the large (60S) subunit . The final stages of ribosomal subunit maturation follow the export of pre-ribosomal particles to the cytoplasm, forming the active 40S and 60S subunits of eukaryotic ribosomes .

45. Ribosomal proteins are imported to the nucleolus from the cytoplasm and begin to assemble on pre-rRNA prior to its cleavage. As the pre-rRNA is processed, additional ribosomal proteins and the 55 rRNA (which is synthesized elsewhere in the nucleus) assemble to form preribosomal particles. The final steps of maturation follow the export of preribosomal particles to the cytoplasm, yielding the 405 and 605 ribosomal subunits. Ribosome assembly

46. Thank You

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