Translational Regulation

1
Translational Regulation

• Sunnie Thompson
• PTRM 08/26/08

2
Eukaryotic and Prokaryotic Translation
Differences

1. Translation and Transcription are uncoupled.
2. mRNAs are monocistronic.
3. Ribosomes bind to the RNA at the 5end (no Shine
   Dalgarno sequence).
4. mRNA has a 5cap structure.
5. mRNA has a poly(A) tail (except Histone mRNA)
6. tRNAmeti is specific for initiation in
   eukaryotes it is not a formylated tRNAmet

Similarities

1. The genetic code is nearly universal, applying to
   all species on our planet. Protein synthesis
   begins with an AUG and terminates with UGA, UAA,
   and UAG.

3
Differences in Translational Complexity Between
Prokaryotes and Eukaryotes
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Summary
Mechanisms of initiation Cap-dependent
translation Cap-independent translation
Internal ribosome entry sites (IRES) Regulation
of initiation Global eIF2 alpha
phosphorylation 4E-BPs Cleavage of
eIF4G Message specific IRES Derepression
during starvation GCN4 Steric blockage
IRE/IRP Cap-dependent CPEB/Maskin Cap-indepen
dent SXL Post-recruitment hnRNPK/hnRNPE1 Loc
alization ASH1 miRNA




5
mRNA

• Cap
• Poly(A) tail
• Secondary structures
• IRES
• uORF
• Protein/RNA binding sites
• AUG Kozak consensus sequence GCC(A/G)CCAUGG

Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
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Scanning model of Cap-dependent Translation
Initiation
eIF4F eIF4E cap binding protein eIF4G
Scaffolding protein eIF4A Helicase

1. Ternary complex formation
2. 43S pre-initiation complex
3. Recruitment to the 5end of the mRNA

E
P
A
eIF5B
Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
E
P
A
7
Closed-loop model

http//departments.oxy.edu/biology/Stillman/bi221/
091300/091300_lecture_figures.htm
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Scanning model of Cap-dependent Translation
Initiation

1. Ternary complex formation
2. 43S pre-initiation complex
3. eIF4F recruits 43S to the 5end of the mRNA
   geneerating 48S complex
4. 43S complex scans down to the AUG
5. eIF2 positions the met-tRNA in the P-site of the
   ribosome. eIF1 aids in correct start codon
   selection
6. eIF5 hydrolyses eIF2-GTP, release of initiation
   factors
7. eIF1A recruits eIF5B-GTP
8. 60S subunit joins
9. Hydrolysis of eIF5B-GTP and release of eIF1A and
   5B

E
P
A
eIF5B
Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
E
P
A
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Regulation of Cap-dependent Translation Initiation
X

• Reduction of Ternary complex
• Inhibition of eIF4E binding to the cap
• Cleavage of eIF4G
• Inhibition of 43S recruitment
• Block 60S subunit joining

X
X
X
E
P
A
eIF5B
X
Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
E
P
A
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Polysome Analysis

Polysomes

Actin Northern
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Global Regulation
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Recycling of the ternary complex requires eIF2B
Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
13
eIF2? Phosphorylation Reduces Ternary Complex
Starvation (GCN2) Viral infection, apoptisis
(PKR) ER Stress (PERK) Haemin-regulated inhibitor
(HRI)
Fátima Gebauer Matthias W. Hentze Nature
Reviews Molecular Cell Biology 5, 827-835 (2004)
14
GCN4 expression is regulated by uORFs
uORFS
GCN4
1
2
3
4

• Encodes a transcriptional activator of genes that
  regulate AA biosynthesis
• Not expressed when AA are abundant
• Translation is induced when AA are depleted
• The scanning model predicts that the first AUG
  will be recognized and translated
• Mis-sense mutations had no effect so it unlikely
  that uORFs functioned as a sensor for AA
  starvation

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uORFs function in GCN4 translational control
Only need uORF 1 and 4 for regulation
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uORFs function in GCN4 translational control
Only need uORF 1 and 4 for regulation
Removing all uORFs results in constitutive
expression of GCN4
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uORFs function in GCN4 translational control
Only need uORF 1 and 4 for regulation
Removing all uORFs results in constitutive
expression of GCN4
Removing uORF1 resulted in no expression under
starvation conditions
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uORFs function in GCN4 translational control
Only need uORF 1 and 4 for regulation
Removing all uORFs results in constitutive
expression of GCN4
Removing uORF1 resulted in no expression under
starvation conditions
Removing uORF4 results in constitutive expression
of GCN4
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Abundance of Ternary complex regulates GCN4
Non-starvation
Starvation
F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827
20
eIF4E binding proteins 4E-BP
4E-BP eIF4E binding proteins
Apoptosis Hypoxia

Insulin AA (Leucine) Cell Proliferation
F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827
4E-T (eIF4E transporter) inhibits translation and
promotes P-body formation
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Cleavage of eIF4G
SJ Morley, MJ Codwell, MJ Clemens (2006) Cell
Death and Diff. 12, 571-584.
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Message specific regulation of translation
initiation
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Viral IRES

• XYgt IRES Trans-acting factors

Stoneley and Willis (2004) Oncogene 23, 3200-3207
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Mechanisms of Internal Initiation
IRES

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Dicistronic Assay for IRES activity
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Cap-Independent Translation Initiation

• Cellular
• Cellular stress viral infection (PKR),
  apoptosis, hypoxia
• Normal cellular processes G2/M phase cell cycle
• Viral
• Genomes Picornaviruses, Hepatitis C Virus,
  Cricket paralysis virus
• Specific viral messages HIV, Herpes virus (.
  and the list is still growing!)

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Cellular IRESs How do they recruit 40S?

• Widely assumed that the Secondary and tertiary
  Structure allow for interactions with
  translational machinery (ITAF,eIFs, 40S)
• No common Structure
• Composed of multiple short modules
• Generally 150-300 nts long, exception 9nt
  repeated element.

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mRNA-rRNA base-pairing
Dresios et al. (2006) Nature Struct. Mol. Bio.
13, 30-34.
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IRE/IRP steric blockage

• Binding of the IRP to the IRE sterically hinders
  the ability of the 43S initiation complex from
  associating with the mRNA, although eIF4F is
  bound to the mRNA.
• The position of the IRE to the cap is essential
  for regulation
• Another RNA SL and binding partner can
  functionally replace IRE/IRP

F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827
30
Maskin and Bicoid are mRNA specific 4E-BP
(Cap-dependent repression)

• CPEB binds Maskin which binds eIF4E and prevents
  eIF4G binding
• Bicoid binds both BRE and eIF4E to repress caudal
  mRNA

F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827
31
Cap-independent regulation

• SXL binds a U-rich sequence in the 5UTR and
  3UTR
• SXL recruits UNR, a co-repressor, that inhibits
  the association of the 43S complex
• 5UTR bound SXL blocks any scanning by the 43S
  complex that may have escaped the
• SXL-UNR blockage

Ann-Bin Shyu. Nature Struct. Molec. Biol.
(2006) 13, 189-190.
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Post-recruitment hnRNPK/hnRNPE1

• Independent of Poly(A) and Cap
• Sucrose gradient 48S complex
• Toe-printing 43S complex at the initiator AUG
• hnRNP EI and K prevent 60S joining

F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827
33
Toe-print assay
Method used to determine the position of a 43S or
80S complex on an mRNA.
F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol
5, 827 (Oct, 2004).
RT

1. A complex is formed (48S or 80S)
2. An oligo is annealed downstream
3. Reverse transcription reaction
4. Analysis of RT stops on a sequencing gel
5. If the AUG is in the P-site of the ribosome
   expect a stop at 16-18 downstream

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Lox mRNA toe-print
D. H. Ostareck, A. Ostareck-Lederer, I. N.
Shatsky, M. W. Hentze, Cell 104, 281
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Localization

• mRNA must be translationally silent during
  transport (Puf6p and Khd1p)
• mRNA is packaged in locasomes for transport
  along actin bundles
• Zipcodes lie within the message (typically in
  the 3UTR)
• Anchoring of the message at the site of
  localization
• Translational activation (phosphorylation of
  repressors)

N. Paquin, P. Chartrand, Trends Cell Biol 18, 105
(Mar, 2008).
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Summary
Mechanisms of initiation Cap-dependent
translation Cap-independent translation
Internal ribosome entry sites (IRES) Regulation
of initiation Global eIF2 alpha
phosphorylation 4E-BPs Cleavage of
eIF4G Message specific IRES Derepression
during starvation GCN4 Steric blockage
IRE/IRP Cap-dependent CPEB/Maskin Cap-indepen
dent SXL Post-recruitment hnRNPK/hnRNPE1 Loc
alization ASH1 miRNA
37
(No Transcript)