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Chapter 5.

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Presentation on theme: "Chapter 5."— Presentation transcript:

1 Chapter 5

2 Figure 9.2: The nucleus is delimited by the nuclear envelope.
Introduction The nucleus contains most of the cell’s DNA, allowing for sophisticated regulation of gene expression. The nuclear envelope is a double membrane that surrounds the nucleus. Figure 9.2: The nucleus is delimited by the nuclear envelope. Photo courtesy of Terry Allen, University of Manchester.

3 Introduction The nucleus contains subcompartments that are not membrane-bounded. The nuclear envelope contains pores used for: passive diffusion of small molecules (sugars, nucleotides, amino acids) actively importing proteins into the nucleus actively exporting RNAs and proteins from the nucleus

4 Introduction

5 Nuclei vary in appearance according to cell type and organism
Nuclei range in size from about one micron (1 μm) to more than 10 μm in diameter. Most cells have a single nucleus, but some cells contain multiple nuclei, and a few cell types lack nuclei. The percentage of the genome that is heterochromatin varies among cells and increases as cells become more differentiated.

6 Chromosomes occupy distinct territories
Although the nucleus lacks internal membranes, nuclei are highly organized and contain many subcompartments. Each chromosome occupies a distinct region or territory which prevents chromosomes from becoming entangled with one another.

7 Chromosomes occupy distinct territories
The nucleus contains both chromosome domains and interchromosomal regions. Figure 9.06: Individual chromosomes occupy distinct areas of the nucleus called chromosome territories. Photo reproduced with permission from J. Cell Sci., vol. 114 (16): [ Photo courtesy of Thomas Reid, National Institutes of Health.

8 The nucleus contains subcompartments that are not membrane-bounded
Nuclear subcompartments are not membrane-bounded. rRNA is synthesized and ribosomal subunits are assembled in the nucleolus. The nucleolus contains DNA that encodes rRNAs and that is present on multiple chromosomes.

9 The nucleus contains subcompartments that are not membrane-bounded
mRNA splicing factors: are stored in nuclear speckles move to sites of transcription where they function Other nuclear bodies can be identified with antibodies, but the functions of most of these are unknown. Figure 9.8: Splicing factors are stored in nuclear speckles. Photos courtesy of David Spector, Cold Spring Harbor Laboratory.

10 Some processes occur at distinct nuclear sites and may reflect an underlying structure
The nucleus contains replication site, sometimes called replication factories, where DNA is synthesized. The nucleus may contain a nucleoskeleton that could help to organize nuclear functions. Figure 9.11: Enzymatic machines that replicate DNA and splice RNA may be anchored to a nuclear matrix.

11 The nucleus is bounded by the nuclear envelope
The nucleus is surrounded by a nuclear envelope consisting of two complete membranes. The outer nuclear membrane is continuous with the membranes of the endoplasmic reticulum (ER) and the lumen of the nuclear envelope is continuous with the lumen of the ER. The nuclear envelope contains numerous NPCs, the only channels for transport of molecules and macromolecules between the nucleus and the cytoplasm.

12 The nucleus is bounded by the nuclear envelope
Figure 9.12: The nuclear envelope is continuous with the ER. Photo courtesy of Terry Allen, University of Manchester.

13 The nuclear lamina underlies the nuclear envelope
The nuclear lamina is constructed of intermediate filament proteins called lamins. The nuclear lamina is located beneath the inner nuclear membrane and is physically connected to it by lamina-associated integral membrane proteins. The nuclear lamina plays a role in nuclear envelope assembly and may provide physical support for the nuclear envelope. Proteins connect the nuclear lamina to chromatin; this may allow the nuclear lamina to organize DNA replication and transcription.

14 The nuclear lamina underlies the nuclear envelope
Protein complexes that interact with the nuclear lamina cross the nuclear envelope and link the cytoskeleton to the nuclear interior. Yeast and some other unicellular eukaryotes lack a nuclear lamina. Figure 9.16: The nuclear lamina is anchored to the inner nuclear envelopes and NPCs.

15 Large molecules are actively transported between the nucleus and cytoplasm
Uncharged molecules smaller than 100 daltons (da) can pass through the membranes of the nuclear envelope. Molecules and macromolecules larger than 100 daltons (da) cross the nuclear envelope by moving through NPCs. Figure 9.18: Many different classes of molecules and macromolecules are transported through NPCs.

16 Large molecules are actively transported between the nucleus and cytoplasm
Particles up to 9 nm in diameter (corresponding to globular proteins up to 40 kDa) can pass through NPCs by passive diffusion, as can metabolites, nucleotides, and other small molecules. Larger macromolecules are actively transported through NPCs and must contain specific information in order to be transported. Figure 9.19: When polvinylo-pyrollidine-coated gold particles of various sizes are injected into cells, those smaller than 9 nm can pass through NPDs by passive diffusion.

17 Nuclear pore complexes are symmetrical channels
NPCs are symmetrical structures that are found at sites where the inner and outer nuclear membrane are fused. Each NPC in human cells has a mass of ~ megadaltons which is 40 times that of a ribosome, and is constructed from multiple copies of about 30 proteins.

18 Nuclear pore complexes are symmetrical channels
NPCs contain fibrils that extend into the cytoplasm and a basket-like structure that extends into the nucleus. Figure 9.24: Inner and outer nuclear membranes of the nuclear envelope are fused at the NPC.

19 Nuclear pore complexes are constructed from nucleoporins
The proteins of NPCs are called nucleoporins. Many nucleoporins contain repeats of short sequences, which are thought to interact with transport factors during transport such as Gly-Leu-Phe-Gly (GLFG), X-Phe-X-Phe-Gly (XFXFG) and X-X-Phe-Gly (XXFG), which are believed to interact with transport factors during the transport.

20 Nuclear pore complexes are constructed from nucleoporins
Some nucleoporins are transmembrane proteins that are thought to anchor NPCs in the nuclear envelope. All of the nucleoporins of yeast NPCs and most if not all nucleoporins of mammalian NPCs have been identified. NPCs are diassembled and reassembled during mitosis in cells undergoing an open mitosis. Some nucleoporins are dynamic: they rapidly associate with and dissociate from NPCs. Others are stable.

21 Proteins are selectively transported into the nucleus through nuclear pores
Mature nuclear proteins contain sequence information required for their nuclear localization. Proteins selectively enter and exit the nucleus through nuclear pores. Information for nuclear import lies in a small portion of the transported protein. Figure 9.30: The rates of import and export for a few of the macromolecules that are transported through nuclear pores. Rates reflect primarily the relative amount of each protein present and transported.

22 Nuclear localization sequences target proteins to the nucleus
A nuclear localization sequence (NLS) is often a short stretch of basic amino acids. NLSs are defined as sequences both necessary and sufficient for nuclear import. Figure 9.32: NLSs contain short stretches of basic amino acids. Photos reprinted from Cell, vol. 39, D. Kalderon, et al., A short amino acid sequence..., pp , Copyright (1984) with permission from Elsevier [ Photo courtesy of Daniel Kalderon, Columbia University.

23 Cytoplasmic receptors recognize nuclear localization sequence (NLSs) and mediate nuclear protein import Receptors for nuclear import are cytoplasmic proteins that bind to the NLSs of cargo proteins. Nuclear import receptors are part of a large family of proteins often called karyopherins. Karyopherins is the name given to the group of proteins that play roles in import of proteins and some RNAs from the nucleus. Most are involved either to import or in export and are often referred to as importins and exportins, respectively. Figure 9.35: Many cargo proteins are imported by binding directly to karyopherins.

24 Export of proteins from the nucleus is also receptor-mediated
Short stretches of amino acids rich in leucine act as the most common type of nuclear export sequence (NES). A nuclear export receptors binds proteins that contain NESs in the nucleus and transports them to the cytoplasm.

25 Export of proteins from the nucleus is also receptor-mediated
Some proteins, such as HIV Rev, shuttle between the nucleus and the cytoplasm. For simplicity, other proteins involved in transport are not shown. Figure 9.36: Nucleocytoplasmic shutting of HIV Rev protein.

26 The Ran GTPase controls the directionality and irreversibility of nuclear transport
Ran is a small GTPase that is common to all eukaryotes and is found in both the nucleus and the cytoplasm. The Ran-GAP (GTP-ase activating protein) promotes hydrolysis of GTP by Ran , whereas Ran-GEF (guanine-nucleotide exchange factor) promotes exchange of GDP for GTP on Ran.

27 Ran GTPase The Ran-GAP is cytoplasmic, whereas the Ran-GEF is located in the nucleus and is associated with chromatin. Ran controls nuclear transport by binding karyopherins and affecting their ability to bind their cargoes. Figure 9.40: Importins bind cargos in the cytoplasm and release them in the nucleus after binding Ran-GTP. Conversely, export complexes form in the nucleus together with RAN-GTP.

28 Multiple models have been proposed for the mechanism of nuclear transport
Interactions between karyopherins and nucleoporins are critical for translocation across the nuclear pore. Directionality may be conferred in part by distinct interactions of karyopherins with certain nucleoporins. Figure 9.42: The contacts between karyopherins and nucleoporins via the phenylalanine-glycine repeats (FG repeats).

29 Nuclear transport can be regulated
Both protein import and export can be regulated. Cells use nuclear transport to regulate many functions, including: transit through the cell cycle and response to external stimuli. The localization of the transcription factor NF-KB illustrates how nuclear transport is regulated. Figure 9.44: NF - κB moves into and out of the nucleus in a highly regulated manner.

30 Multiple classes of RNA are exported from the nucleus
mRNAs, tRNAs, and ribosomal subunits produced in the nucleus are exported through NPCs to function during translation in the cytoplasm. Figure 9.45: Most RNAs are exported from the nucleus.

31 Multiple classes of RNA are exported from the nucleus
The same NPCs used for protein transport are also used for RNA export. Export of RNA is also receptor-mediated and energy-dependent. Different soluble transport factors are required for transport of each class of RNA.

32 Ribosomal subunits are assembled in the nucleolus and exported by multiple receptors
Ribosomal subunits are assembled in the nucleolus where rRNA is synthesized. Ribosomal proteins are imported from the cytoplasm for assembly into the ribosomal subunits. Export of the ribosomal subunits is carrier-mediated and requires Ran.

33 Figure 9.49: Assembly and export of ribosomal subunits.
tRNAs are exported by a dedicated exportin and can also use other receptors Exportin-t, is the primary transport receptor for tRNAs, but other receptors and exportins can also export tRNAs. tRNA export is affected by modifications of the tRNAs. tRNAs can be imported back into the nucleus and subsequently reexported. Both exportins and aminoacl tRNA synthetase function as export receptors for final export. Figure 9.49: Assembly and export of ribosomal subunits.

34 mRNAs are exported from the nucleus as RNA-protein complexes
Proteins that associate with mRNAs during transcription help to define sites of pre-mRNA processing and are also believed to package mRNAs for export.

35 mRNAs are exported from the nucleus as RNA-protein complexes
Most proteins that associate with mRNA in the nucleus are removed after export and returned to the nucleus. A few are removed immediately prior to export. Signals for mRNA export may be present in proteins bound to the mRNA. The export of mRNA can be regulated, but the mechanism for this is unknown.

36 hnRNPs move from sites of processing to nuclear pore complexes
mRNAs are released from chromosome territories into interchromosomal domains following completion of pre-mRNA processing. mRNAs move to the nuclear periphery by diffusion through interchromosomal spaces.

37 mRNA export requires several novel factors
Many factors required uniquely for mRNA export have been identified. Factors able to bind to both the mRNP and NPC help to mediate mRNA export. Figure 9.54: TAP binds to both mRNPs and to NPCs and functions as an export receptor for mRNA.

38 mRNA export requires several novel factors
One factor, Dbp5, is an ATPase and may use energy from ATP hydrolysis to dissociate mRNP proteins from the mRNA during export. Figure 9.55: Dbp5 associates with the terminal filaments of the NPC where it interacts with Gle1/inositol hexakisphosphate and harness energy from ATP hydrolysis to remove mRNP proteins.

39 Figure 9.56: Formation of snRNPs.
U snRNAs are exported, modified, assembled into complexes, and imported back into the nucleus In most organisms, U snRNAs produced in the nucleus are exported, modified, packaged into U snRNP RNA-protein complexes, imported into the nucleus and processed further into mature U snRNPs, which function in RNA processing Figure 9.56: Formation of snRNPs.

40 Figure 9.57: Model for generating mature mRNA.
Precursors to microRNAs are partially processed in the nucleus, exported and further processed in the cytoplasm MicroRNAs are produced by transcription in the nucleus, partial processing to generate a hairpin precursor, export of the precursor by exportin-5, and final processing in the cytoplasm. Figure 9.57: Model for generating mature mRNA.


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