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Chapter 5

Chapter 5. Nuclear structure and transport By Charles N. Cole & Pamela A. Silver. 5.1 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. 5.1 Introduction.

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Chapter 5

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  1. Chapter 5 Nuclear structure and transport By Charles N. Cole & Pamela A. Silver

  2. 5.1 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.

  3. 5.1 Introduction • The nucleus contains subcompartments that are not membrane-bounded. • The nuclear envelope contains pores used for: • importing proteins into the nucleus • exporting RNAs and proteins from the nucleus

  4. 5.2 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.

  5. 5.3 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. • This prevents chromosomes from becoming entangled with one another.

  6. 5.3 Chromosomes occupy distinct territories • The nucleus contains both chromosome domains and interchromosomal regions.

  7. 5.4 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.

  8. 5.4 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.

  9. 5.5 Some processes occur at distinct nuclear sites and may reflect an underlying structure • The nucleus contains replication sites where DNA is synthesized. • The nucleus may contain a nucleoskeleton that could help to organize nuclear functions.

  10. 5.6 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). • The lumen of the nuclear envelope is continuous with the lumen of the ER. • The nuclear envelope contains numerous NPCs. • They are the only channels for transport of molecules and macromolecules between the nucleus and the cytoplasm.

  11. 5.7 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. • They are physically connected 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.

  12. 5.7 The nuclear lamina underlies the nuclear envelope • Proteins connect the nuclear lamina to chromatin; • this may allow the nuclear lamina to organize DNA replication and transcription. • Yeast and some other unicellular eukaryotes lack a nuclear lamina.

  13. 5.8 Large molecules are actively transported between the nucleus and cytoplasm • Uncharged molecules smaller than 100 daltons can pass through the membranes of the nuclear envelope. • Molecules and macromolecules larger than 100 daltons cross the nuclear envelope by moving through NPCs.

  14. 5.8 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. • Larger macromolecules are actively transported through NPCs and must contain specific information in order to be transported.

  15. 5.9 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 ~120 106 daltons (40 times that of a ribosome). • It is constructed from multiple copies of ~30 proteins.

  16. 5.9 Nuclear pore complexes are symmetrical channels • NPCs contain: • fibrils that extend into the cytoplasm • a basket-like structure that extends into the nucleus

  17. 5.10 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 • X-Phe-X-Phe-Gly • X-X-Phe-Gly

  18. 5.10 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 have been identified. • NPCs are disassembled and reassembled during mitosis. • Some nucleoporins are dynamic: they rapidly associate with and dissociate from NPCs.

  19. 5.11 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.

  20. 5.12 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 both necessary and sufficient for nuclear import.

  21. 5.13 Cytoplasmic NLS receptors mediate nuclear protein import • Receptors for nuclear import are cytoplasmic proteins that bind to the NLS of cargo proteins. • Nuclear import receptors are part of a large family of proteins often called karyopherins.

  22. 5.14 Export of proteins from the nucleus is also receptor-mediated • Short stretches of amino acids rich in leucine act as the most common nuclear export sequences. • A nuclear export receptor: • binds proteins that contain nuclear export sequences (NESs) in the nucleus • transports them to the cytoplasm

  23. 5.15 The Ran GTPase controls the direction 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 promotes hydrolysis of GTP by Ran. • The Ran-GEF promotes exchange of GDP for GTP on Ran.

  24. 5.15 The Ran GTPase controls the direction of nuclear transport • The Ran-GAP is cytoplasmic, whereas the Ran-GEF is located in the nucleus. • Ran controls nuclear transport by binding karyopherins and affecting their ability to bind their cargoes.

  25. 5.16 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.

  26. 5.17 Nuclear transport can be regulated • Both protein import and export are regulated. • Cells use nuclear transport to regulate many functions, including: • transit through the cell cycle • response to external stimuli • The movement of the transcription factor NF-κB illustrates how nuclear transport is regulated.

  27. 5.18 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.

  28. 5.18 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 receptor-mediated and energy-dependent. • Different soluble transport factors are required for transport of each class of RNA.

  29. 5.19 Ribosomal subunits are assembled in the nucleolus and exported by exportin 1 • Ribosomal subunits are assembled in the nucleolus where rRNA is made. • Ribosomal proteins are imported from the cytoplasm for assembly into the ribosomal subunits. • Export of the ribosomal subunits is carrier-mediated and requires Ran.

  30. 5.20 tRNAs are exported by a dedicated exportin • Exportin-t is the transport receptor for tRNAs. • tRNA export requires Ran. • tRNA export may be affected by modifications of the tRNAs. • tRNAs may be re-imported into the nucleus.

  31. 5.21 Messenger RNAs are exported from the nucleus as RNA-protein complexes • Proteins that associate with mRNAs during transcription help to define sites of pre-mRNA processing. • They are also thought to package mRNAs for export.

  32. 5.21 Messenger RNAs 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.

  33. 5.22 hnRNPs move from sites of processing to NPCs • 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.

  34. 5.23 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 nuclear pore complex help to mediate mRNA export.

  35. 5.23 mRNA export requires several novel factors • One factor, Dbp5, is an ATPase and may use energy from ATP hydrolysis to remove mRNP proteins during transport.

  36. 5.24 U snRNAs are exported, modified, assembled into complexes, and imported • U snRNAs produced in the nucleus are • exported • modified • packaged into U snRNP RNA-protein complexes • imported into the nucleus to function in RNA processing

  37. 5.25 Precursors to microRNAs are exported from the nucleus and 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-V • final processing in the cytoplasm

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