1. The Nucleus 10

2. 10 The Nucleus The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The Organization of Chromosomes Nuclear Bodies

3. Introduction The nucleus is the main feature that distinguishes eukaryotic from prokaryotic cells. It houses the genome, and thus is the repository of genetic information and the cell’s control center. Separation of the genome from the site of mRNA translation plays a central role in eukaryotic gene expression.

4. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The nuclear envelope separates the nuclear contents from the cytoplasm. It controls traffic of proteins and RNAs through nuclear pore complexes, and plays a critical role in regulating gene expression.

5. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The nuclear envelope consists of: Two nuclear membranes An underlying nuclear lamina Nuclear pore complexes

6. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The outer membrane is continuous with the endoplasmic reticulum (ER). The space between inner and outer membranes is directly connected with the lumen of the ER. The inner membrane has integral proteins, including ones that bind the nuclear lamina.

7. Figure 10.1 The nuclear envelope (Part 1)

8. Figure 10.1 The nuclear envelope (Part 2)

9. Figure 10.1 The nuclear envelope (Part 3)

10. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Nuclear membranes are phospholipid bilayers permeable only to small nonpolar molecules. Nuclear pore complexes are the only channels for small polar molecules, ions, and macromolecules.

11. Figure 10.2 Electron micrograph showing nuclear pores

12. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Nuclear lamina: a fibrous mesh that provides structural support. Consists of fibrous proteins (lamins) and other proteins.

13. Figure 10.3 Electron micrograph of the nuclear lamina

14. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Lamins are a class of intermediate filament proteins that associate to form higher order structures. Two lamins interact to form a dimer: the α -helical regions wind around each other to form a coiled coil. The lamin dimers associate with each other to form the lamina.

15. Figure 10.4 Lamin assembly

16. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Lamins bind to inner membrane proteins such as emerin and lamin B receptor (LBR). They are connected to the cytoskeleton by LINC protein complexes. Lamins also bind to chromatin.

17. Figure 10.5 The nuclear lamina

18. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Mutations in lamin genes cause several inherited tissue-specific diseases. The bases of the pathologies in each of these diseases is still unclear.

19. Molecular Medicine, Ch. 10, p. 371 (Part 1)

20. Molecular Medicine, Ch. 10, p. 371 (Part 2)

21. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Nuclear pore complexes are large; about 30 proteins (nucleoporins). RNAs synthesized in the nucleus must be exported to the cytoplasm for protein synthesis. Proteins needed for nuclear functions must be imported from synthesis sites in the cytoplasm.

22. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Molecules pass through pore complexes by two mechanisms: Passive diffusion—small molecules pass freely in either direction. Proteins and RNAs are selectively transported; requires energy.

23. Figure 10.6 Molecular traffic through nuclear pore complexes

24. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Electron microscopy shows pore complexes have eight subunits organized around a large central channel.

25. Figure 10.7 Electron micrograph of nuclear pore complexes

26. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Eight spokes are connected to rings at the nuclear and cytoplasmic surfaces. The spoke-ring assembly surrounds a central channel. Protein filaments extend from the rings, forming a basketlike structure on the nuclear side.

27. Figure 10.8 Model of the nuclear pore complex

28. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Proteins that must enter the nucleus have amino acid sequences called nuclear localization signals. These signals are recognized by nuclear transport receptors.

29. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Nuclear localization signals were first identified in 1984, using a viral replication protein SV40 T antigen. The amino acid sequence responsible for nuclear localization was determined using T antigen mutants. When the same sequence was attached to other proteins, they were also transported to the nucleus.

30. Key Experiment, Ch. 10, p. 374 (2)

31. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The T antigen nuclear localization signal is a single stretch of amino acids. Other signals are bipartite: two amino acids sequences are separated by another amino acid sequence.

32. Figure 10.9 Nuclear localization signals

33. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Nuclear localization signals (NLS) are recognized by receptors called importins, which carry proteins through the nuclear pore complex. Importins work in conjunction with the GTP-binding protein Ran, which controls directionality of movement.

34. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Importins bind to the NLS of a protein, then to nuclear pore proteins and the complex is transported across the membrane. Ran/GTP binds to the importin, and this complex is transported back. In the cytoplasm, Ran GAP hydrolyzes the GTP on Ran to GDP, releasing the importin.

35. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm The Ran/GDP formed in the cytoplasm is then transported back to the nucleus by its own import receptor, where Ran/GTP is regenerated.

36. Figure 10.10 Protein import through the nuclear pore complex

37. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Protein export from the nucleus: Proteins are targeted for export by amino acid sequences called nuclear export signals (NES). NES are recognized by receptors in the nucleus (exportins), which direct protein transport to the cytoplasm.

38. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Many importins and exportins are members of a family of nuclear transport receptors known as karyopherins.

39. Table 10.1 Examples of Karyopherins

40. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Exportins form stable complexes with cargo proteins in association with Ran/GTP in the nucleus. In the cytoplasm, GTP hydrolysis and release of Ran/GDP leads to dissociation of the cargo protein.

41. Figure 10.11 Nuclear export

42. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm RNAs are transported to the cytoplasm as ribonucleoprotein complexes (RNPs). Karyopherin exportins transport tRNAs, rRNAs, miRNAs.

43. Figure 10.12 Transport of a ribonucleoprotein complex (Part 1)

44. Figure 10.12 Transport of a ribonucleoprotein complex (Part 2)

45. Figure 10.12 Transport of a ribonucleoprotein complex (Part 3)

46. Figure 10.12 Transport of a ribonucleoprotein complex (Part 4)

47. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm mRNA transport does not involve karyopherins and is independent of Ran. A distinct transporter complex moves the mRNA through the nuclear pore. Helicase on the cytoplasm side releases the mRNA and ensures unidirectional transport.

48. Figure 10.13 mRNA export

49. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Many noncoding RNAs (snRNAs and snoRNAs) function within the nucleus. snRNAs are initially exported from the nucleus by an exportin (Crm1). In the cytoplasm, the snRNAs associate with proteins to form snRNPs, which are recognized by an importin and transported back to the nucleus.

50. Figure 10.14 Transport of snRNAs between nucleus and cytoplasm

51. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Regulation of protein transport is a mechanism for controlling protein activity in the nucleus. Example: Regulation of import and export of transcription factors is a way of controlling gene expression.

52. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm In one mechanism, transcription factors or other proteins associate with cytoplasmic proteins that mask their NLS, and so they remain in the cytoplasm.

53. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Transcription factor NF-kB is complexed with IkB in the cytoplasm. If IkB is phosphorylated and degraded by ubiquitin-mediated proteolysis, NF- kB can enter the nucleus and activate transcription of its target genes.

54. Figure 10.15 Regulation of nuclear import of transcription factors

55. The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm Other transcription factors are regulated directly by phosphorylation. Example: Yeast transcription factor Pho4 is phosphorylated at a site adjacent to its NLS, which interferes with its import.

56. The Organization of Chromosomes Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes. During interphase, most of the chromatin decondenses and is distributed throughout the nucleus.

57. The Organization of Chromosomes But even in interphase, the chromosomes occupy distinct regions and are organized such that transcriptional activity of a gene is correlated with its position. DNA replication and transcription take place in clustered regions within the nucleus.

58. The Organization of Chromosomes This organization was first suggested in 1885 and confirmed in 1984 by studies of polytene chromosomes in Drosophila salivary glands. Each chromosome was found to occupy a discrete region of the nucleus, called a chromosome territory.

59. Figure 10.16 Chromosome organization (Part 1)

60. Figure 10.16 Chromosome organization (Part 2)

61. Figure 10.17 Organization of Drosophila chromosomes (Part 1)

62. Figure 10.17 Organization of Drosophila chromosomes (Part 2)

63. The Organization of Chromosomes In situ hybridization with fluorescent probes specific for repeated sequences on individual chromosomes has been used to visualize the location of chromosomes within a nucleus.

64. Figure 10.18 Organization of chromosomes in the mammalian nucleus

65. The Organization of Chromosomes In living cells, chromosome conformation capture (3C) techniques reveal sites of interactions between chromosomal regions. They are identified by cross-linking interacting DNA sequences, which are then amplified and identified by high- throughput sequencing.

66. The Organization of Chromosomes In interphase cells, the euchromatin is decondensed and transcriptionally- active, and is distributed throughout the nucleus. Heterochromatin is highly condensed and not transcribed, and is often associated with the nuclear envelope or periphery of the nucleolus.

67. Figure 10.19 Heterochromatin in interphase nuclei

68. The Organization of Chromosomes Some human chromosomes are rich in transcribed genes, whereas others contain relatively few genes. Fluorescent in situ hybridization shows that gene-rich chromosomes are located in the center of the nucleus; gene-poor chromosomes are at the periphery.

69. Figure 10.20 Fluorescent in situ hybridization to transcriptionally-active and-inactive chromosomes (Part 1)

70. Figure 10.20 Fluorescent in situ hybridization to transcriptionally-active and-inactive chromosomes (Part 2)

71. The Organization of Chromosomes Genomes are divided into topologically associating domains (TADs). Regions within a domain interact frequently with one another, but only rarely with regions in other domains.

72. Figure 8.20 Chromosomal domains and CTCF

73. The Organization of Chromosomes Some domains are associated with the nuclear lamina (called lamina- associated domains or LADs). The genes within LADs are generally transcriptionally repressed. LADS correspond to heterochromatin.

74. Figure 10.21 Distribution of transcriptionally-active and -inactive chromatin

75. The Organization of Chromosomes The nucleolus is also surrounded by heterochromatin (called nucleolus- associated domains or NADs). DNA sequences found in NADs substantially overlap with those in LADs.

76. The Organization of Chromosomes Most nuclear processes occur in distinct regions. DNA replication takes place in large complexes called replication factories, where replication of multiple DNA molecules takes place.

77. The Organization of Chromosomes These can be seen by labeling cells with bromodeoxyuridine, (an analog of thymidine), then staining with fluorescent antibodies.

78. Figure 10.22 Replication factories (Part 1)

79. Figure 10.22 Replication factories (Part 2)

80. The Organization of Chromosomes Transcription occurs at clustered sites (transcription factories) that contain newly synthesized RNA. Coregulated genes, such as immunoglobulin genes from different chromosomes, may be transcribed in the same factory.

81. Figure 10.23 Transcription factories

82. Nuclear Bodies Nuclear bodies are organelles within the nucleus that concentrate proteins and RNAs for specific processes. They are not enclosed by membranes; they are dynamic structures maintained by protein-protein and protein-RNA interactions.

83. Table 10.2 Examples of Nuclear Bodies

84. Nuclear Bodies The nucleolus functions in rRNA synthesis and ribosome production. Cells need large numbers of ribosomes at specific times for protein synthesis. Actively growing mammal cells have 5 to 10 million ribosomes that must be synthesized each time the cell divides.

85. Nuclear Bodies The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit in the nucleolus by RNA polymerase I, yielding a 45S ribosomal precursor RNA. Transcription of the 5S rRNA takes place outside the nucleolus and is catalyzed by RNA polymerase III.

86. Nuclear Bodies Following each cell division, nucleoli become associated with the nucleolar organizing regions that contain the 5.8S, 18S, and 28S rRNA genes. Transcription of 45S pre-rRNA leads to fusion of small prenucleolar bodies. In most cells, the initially separate nucleoli then fuse to form a single nucleolus.

87. Nuclear Bodies Nucleoli have three regions: fibrillar center, dense fibrillar component, and granular component. They represent sites of progressive stages of rRNA transcription, processing, and ribosome assembly.

88. Figure 10.24 Structure of the nucleolus

89. Nuclear Bodies Each nucleolar organizing region contains a cluster of tandemly repeated rRNA genes separated by spacer DNA. The genes are actively transcribed by RNA polymerase I, and their growing RNA chains can be seen in electron micrographs.

90. Figure 10.25 Ribosomal RNA genes (Part 1)

91. Figure 10.25 Ribosomal RNA genes (Part 2)

92. Nuclear Bodies In higher eukaryotes, the primary transcript of rRNA genes is the 45S pre-rRNA. The pre-rRNA is processed via a series of cleavages, which is similar in all eukaryotes.

93. Figure 10.26 Processing of pre-rRNA

94. Nuclear Bodies Processing of pre-rRNA also includes substantial base modification: Addition of methyl groups to bases and ribose residues, and conversion of uridine to pseudouridine.

95. Nuclear Bodies Nucleoli have over 300 proteins and 200 small nucleolar RNAs (snoRNAs) that function in pre-rRNA processing. snoRNAs are complexed with proteins, forming snoRNPs. They form processing complexes similar to spliceosomes on pre-mRNA.

96. Nuclear Bodies Most snoRNAs guide RNAs to direct specific base modifications of pre-rRNA. Two families of snoRNAs associate with different proteins, which catalyze ribose methylation or pseudouridine formation. snoRNAs have sequences complementary to 18S or 28S rRNA, which include the sites of base modification.

97. Figure 10.27 Role of snoRNAs in base modification of pre-rRNA

98. Nuclear Bodies Formation of ribosomes requires assembly of pre-rRNA with ribosomal proteins and 5S rRNA. Ribosomal proteins are produced in the cytoplasm and imported to the nucleolus, where they assemble with the pre-rRNA prior to cleavage.

99. Nuclear Bodies 5S rRNAs are produced elsewhere in the nucleolus. Additional ribosomal proteins and the 5S rRNA assemble to form pre-ribosomal particles. Pre-ribosomal particles are then exported to the cytoplasm, yielding the 40S and 60S ribosomal subunits.

100. Figure 10.28 Ribosome assembly

101. Nuclear Bodies Polycomb proteins repress transcription of genes via methylation of histone H3 lysine 27 residues. Immunofluorescence shows these proteins are concentrated in domains called Polycomb bodies. Some Polycomb bodies contain clusters of repressed domains from different chromosomal regions.

102. Figure 8.36 Polychrome proteins

103. Figure 10.29 Polycomb bodies (Part 1)

104. Figure 10.29 Polycomb bodies (Part 2)

105. Nuclear Bodies Cajal bodies are involved in assembly of snRNPs and other RNA-protein complexes. snRNAs are modified by ribose methylation and pseudouridylation. The enzyme for RNA methylation (fibrillarin) is concentrated in Cajal bodies.

106. Figure 10.30 Cajal bodies in the nucleus (Part 1)

107. Figure 10.30 Cajal bodies in the nucleus (Part 2)

108. Nuclear Bodies Cajal bodies also have small Cajal body- specific RNAs (scaRNAs). scaRNAs are related to snoRNAs and similarly serve as guides to direct ribose methylation and pseudouridylation of snRNAs.

109. Nuclear Bodies Cajal bodies appear to play a role in assembly of telomerase, which replicates the ends of chromosomal DNA. Cajal bodies may promote assembly of the RNA-protein telomerase complex and facilitate its delivery to telomeres.

110. Nuclear Bodies Following assembly and maturation in Cajal bodies, snRNPs are transferred to speckles, which also contain splicing factors. Speckles are recruited to actively transcribed genes where pre-mRNA processing occurs.

111. Figure 10.31 Nuclear speckles