9 The Nucleus

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9 The Nucleus

• Student Learning Outcomes
• Nucleus distinguishes
• Eukaryotes from Prokaryotes
• Explain general structures of Nuclear Envelope,
  Nuclear Lamina, Nuclear Pore Complex
• Explain movement of proteins and RNA between
  Nucleus and Cytoplasm
• Selective traffic of proteins, RNAs regulates
  gene expression
• Describe the Internal Organization of the
  Nucleus
• Describe the Nucleolus and rRNA Processing

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Nuclear EnvelopeTraffic between Nucleus and
Cytoplasm

• 1. Nuclear envelope
• Two membranes
• Underlying nuclear lamina
• Nuclear pore complexes
• Outer membrane continuous with ER
• membrane proteins bind cytoskeleton
• Inner membrane proteins bind nuclear lamina

Fig. 9.1 EM of nucleus arrows indicate nuclear
pores
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Nuclear membrane, nuclear pores
Fig. 9.1 Outer membrane is continuous with
ER Note ribosomes on ER

• Each nuclear membrane is phospholipid bilayer
  permeable only to small nonpolar molecules.
• Nuclear pore complexes are sole channels for
  small polar molecules, ions, proteins, RNA to
  pass through nuclear envelope.

Fig. 9.2 EM of nucleus arrows indicate nuclear
pores
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Nuclear Envelope,Traffic between Nucleus and
Cytoplasm

• Nuclear lamina is fibrous mesh (structural
  support)
• Fibrous proteins (lamins),
• and other proteins.
• Mutations in lamin genes
• cause inherited diseases

Fig. 9.3 EM of nuclear lamina
Hutchinson-Gilford Progeria causes premature
aging Mutations in LMNA gene affect Lamin A
protein
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Mammals have 3 lamin genes, (A, B, and C), which
  code for at least 7 proteins.
• Two lamins form dimer, a-helical regions of 2
  polypeptide chains wind around each other -gt
  coiled coil.
• Lamin dimers associate to form nuclear lamina.

Fig. 9.4
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Nuclear pore complexes
• large 120 nm
• Complex vertebrates, 30 different proteins
  (nucleoporins)
• Circular structures on faces of membrane 8-fold
  symmetry.
• Lamina loose mesh in nucleus
• Lamins bind
• Protein emerin,
• lamin B receptor (LBR)
• (inner membrane)
• Chromatin.
• .

Figs. 9.5, 9,7
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Nuclear Pore,Traffic between Nucleus and Cytoplasm

• Nuclear pore complex - 8 spokes connected to
  rings at nuclear and cytoplasmic surfaces.
• Spoke-ring assembly
• surrounds central channel
• Protein filaments extend
• from rings
• Basketlike structure
• on nuclear side.
• Cytoplasmic filaments
• on cytoplasmic side

Fig. 9.8 nuclear Pore complex
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Nuclear Pore Complex, Traffic between Nucleus and
Cytoplasm

• Nuclear Pore Complex controls traffic between
• nucleus and cytoplasm
• critical for physiology
• Passive transport
• small molecules pass freely
• through channels
• Selective transport
• energy-dependent
• for macromolecules
• (proteins and RNAs)

Fig. 9.6 nuclear pore complex controls transport
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Nuclear localization signals (NLS)
• Required for proteins to enter nucleus- specific
  aa seq
• Recognized by nuclear transport receptors
• transport of proteins through nuclear pore
• first identified on SV40 T antigen
• (viral replication protein)
• mutants helped figure
• Some NLS are one aa seq
• Others bipartitate seq

A, kinase with SV40 NLS B, mutated NLS
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Import of proteins to nucleus
• NLS recognized by nuclear transport receptors
  importins
• Activity of nuclear transport receptors regulated
  by Ran, a GTP-binding protein
• Importins bind cargo at NLS sequence
• Move through pore
• Ran-GTP unloads, takes importin out.
• High concentration of Ran/GTP in nucleus
• enzyme localization
• GAP does GTP hydrolysis in cytoplasm
• GEF does GDP/ GTP exchange
• in nucleus (Fig. 9.20)

Fig. 9.11 import of proteins
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Nuclear export signals (NES)
• Required for proteins targeted for export
• Signals recognized by exportins
• (receptors in nucleus) direct transport to
  cytoplasm
• Less well characterized than NLS
• Ran also required for nuclear export
• Ran/GTP promotes binding of exportins
• and their cargo proteins,
• Ran/GTP dissociates complexes between importins
  and cargos (see Fig. 9.10)

Fig. 9.12 export of proteins
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Many importins and exportins are family of
nuclear transport receptors - karyopherins.
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Regulation of Protein transport is another point
  at which nuclear protein activity can be
  controlled
• Regulation of import, export of transcription
  factors
• Inhibitors block import (IkB and NF-kB)
• phosphorylation can block import (de-PO4 releases)

Fig. 9.13 regulated import
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Nuclear Envelope, Traffic between Nucleus and
Cytoplasm

• Most RNAs are exported from nucleus to cytoplasm
  to function in protein synthesis
• Active, energy-dependent process
• requires transport receptors
• Transported as ribonucleoprotein
• complexes (RNPs).
• rRNAs associate with ribosomal
• proteins, specific RNA processing
• proteins in nucleolus (Fig. 9.31).
• mRNAs associate with 20 proteins
• during processing, transport

Fig. 9.14 EM of RNP transport insect salivary
gland RNA unfolds
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Fig 9.15 Transport of snRNAs between nucleus and
cytoplasm

• Many small RNAs (snRNAs, snoRNAs) function in
  nucleus.
• snRNAs are transported to cytoplasm by exportin
  (Crm1)
• associate with proteins to form snRNPs and return
  to nucleus snRNPs function in splicing

Fig. 9.15 RNA
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Internal Organization of the Nucleus

• 2. Internal structure of nucleus organized,
  localized
• In animal cells, lamins where chromatin
  attachmes, organize other proteins into
  functional nuclear bodies
• Heterochromatin highly condensed,
• transcriptionally inactive
• Euchromatin decondensed, all over
• Chromosomes organized in territories
• Actively transcribed genes at periphery

Fig. 9.16 arrow nucleolus arrowheads
heterochromatin Fig. 9.19 mammalian nucleus DNA
probes to chrom 4
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Internal Organization of the Nucleus

• Nuclear processes appear localized (sequestered)
  to distinct subnuclear regions
• DNA replication
• Mammalian cells clustered sites labeling newly
  synthesized DNA with bromodeoxyuridine (BrdU in
  place of T)
• Immunofluorescence (Ab to BrdU)
• newly replicated DNA in discrete clusters

Fig. 21 A early replication B, late replication
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Internal Organization of the Nucleus

• Nuclear processes appear localized (sequestered)
  to distinct subnuclear regions
• nuclear speckles mRNA splicing machinery
• Detect with immunofluorescent staining -
  antibodies against snRNPs and splicing factors.
• PML bodies have transcription factors,
  chromatin-modifying proteins identified from
  protein in promyelocytic leukemia

Fig. 9.22 Speckles Fig. 9.23 PML bodies
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The Nucleolus and rRNA Processing

• 3. Nucleolus Site of rRNA transcription,
  processing, some aspects of ribosome assembly.
• Actively growing mammalian cells have 5 to 10 x
  106 ribosomes, must be synthesized each time cell
  divides.
• Nucleolus is not surrounded by a membrane
• Multiple copies of rRNA genes (200 human)
• In oocytes, rRNA genes amplified,
• synthesis for early development.
• rRNA genes amplified 2000-fold in
• Xenopus oocytes, thousands of nucleoli,
• ?1012 ribosomes per oocyte

Fig. 9.26 Xenopus oocyte rRNA genes
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The Nucleolus and rRNA Processing
Fig. 9.28

• Nucleolar
• organizing regions
• After each cell division, nucleoli reform,
  associated to genes for 5.8S, 18S, and 28S rRNA
  genes
• Each nucleolar organizing region has tandemly
  repeated rRNA genes separated by spacer DNA
• 5.8S, 18S, and 28S rRNAs are transcribed as
  single unit in nucleolus by RNA pol I ? 45S
  ribosomal precursor RNA

Fig. 9.25
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Fig 9.29 Processing of pre-rRNA

• Primary transcript of rRNA genes is large 45S
  pre-rRNA
• pre-rRNA processed via series of cleavages, and
  some base modifications, including methylations
• snoRNPs (snoRNAs with proteins) assemble on
  pre-rRNA as processing complexes (like
  spliceosomes on pre-mRNA)

Fig. 9.29 ETS, external transcribed ITS, internal
transcribed
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Fig 9.31 Ribosome assembly

• Formation of ribosomes requires assembly of
  pre-rRNA with ribosomal proteins and 5S rRNA,
  then export of subunits
• pol II made the mRNA for ribosomal proteins.

Fig. 9.31
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• Review questions
• 1. Eukaryote nuclear membranes separate
  transcription from translation. What regulatory
  mechanisms unique to eukaryotes achieve this
  regulation?
• 3. If you inject a frog egg with two globular
  proteins, one 15 kd and the other 100 kd, both of
  which lack NLS, will either protein enter the
  nucleus?
• 4. What determines the directionality of nuclear
  import?
• 5. Describe how the activity of a transcription
  factor can be regulated by nuclear import.
• Consider the effect of mutations at gene level
  that inactivate NLS, NES, prevent phosphorylation
  of key sites, or prevent binding inhibitors on
  function