The Fragile X Protein FMRP Associates with BC1 RNA and Regulates the Translation of Specific mRNAs at Synapses Francesca Zalfa et al. (2003) Cell

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The Fragile X Protein FMRP Associates with BC1
RNA and Regulates the Translation of Specific
mRNAs at SynapsesFrancesca Zalfa et al. (2003)
Cell

• Jeremy Logue

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Dendritic Spines
Nimchinsky, E. et al (2002) Annual Review of
Physiology
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Spine Shape and Plasticity
Nimchinsky, E. et al (2002) Annual Review of
Physiology
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Acitivity Dependent Metamorphosis of Spines
Heike Hering and Morgan Sheng (2001) Nature
Reviews Neuroscience
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Fragile X Syndrome

• Fragile X accounts for about one-half of cases
  of X-linked
• mental retardation and is the most second
  most common
• cause of mental impairment after trisomy 21.
• Caused by a (CGG)n expansion in the FMR1 gene
  promoter.
• Normal individuals harbor 45 /- 25 repeats,
  more than 200
• produces fragile X. Repeats induce
  hypermethylation of the
• promoter, silencing the gene.
• FMR1 encodes fragile X mental retardation
  protein (FMRP),
• an RNA binding protein expressed in the
  brain.
• FRAXA patients and FMR1 KO mice exhibit
  abnormal
• dendritic spines.

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Fragile X Spine Morphology

1. Fragile X patient spines
2. Normal

Irwin, S. et al (2000) Cerebral Cortex
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Macromolecular Structure of the Synapse
Heike Hering and Morgan Sheng (2001) Nature
Reviews Neuroscience
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FMRP Shuttling
Claudia Bagni and William T. Greenough (2005)
Nature Reviews Neuroscience
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FMRP and Synapse Pruning
Claudia Bagni and William T. Greenough (2005)
Nature Reviews Neuroscience
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FMRP Granules
Antar, L et al (2004) J. Neurosci
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mGluR Activation Regulates FMRP Localization
Antar, L et al (2004) J. Neurosci
12
FMRP at Synapses
Claudia Bagni and William T. Greenough (2005)
Nature Reviews Neuroscience
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BC1/BC200 RNA

• BC1 is a 200-nucleotide-long RNA pol III product.
• BC1 RNA is a non-translatable psuedogene.
• BC1 is highly expressed in the brain.
• Composed of three domains
• 5 portion homologous to the Alu Lm
• a central adenosine rich region
• (3) terminal 43-nt non-repetitive domain
• Believed that BC1 was retropositionally generated
  and
• exapted into a function regulating synaptic
  plasticity.

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(No Transcript)
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Polysome/mRNP Distrubution of mRNAs
Sucrose gradient centrifugation of extracts and
RT-PCR for mRNAs. FMR1 KO exhibits shift to
polysome fraction (Note this includes BC1).
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Polysome/mRNP Distribution of mRNAs
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13
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38
Polysome/mRNP analysis
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Protein Levels for FMR1 KO and WT
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Polysome/mRNP Distribution of FMRP Protein
Ribo protein (control)
FMRP co-sediments with monomeric 80S ribosomes
and with mRNPs in total brain and in
synaptoneurosomes.
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mRNAs Associated with FMRP Complex
IP salt dependent
Human
qRT-PCR
Input
KO
KO
KO
KO
wt
wt
wt
wt
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FMRP Binds Directly to BC1 RNA
EMSA using in vitro transcribed BC1 RNA. 750 mM
NaCl used. Concentrations? 25 of RNA
bound. Authors argue that other factors are
required for high-affinity binding.
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Homology Between BC1 RNA and Regulated mRNAs
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BC1 RNA Interacts with FMRP-Targeted mRNAs
21mer DNA oligonucleotide toward region of
complementarity to MAP1B. BC1 RNA hybiridization
to mRNAs is required for FMRP complex formation.
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BC1 RNA Interacts with FMRP-Targeted mRNAs
Total brain RNA mixed with biotin labeled BC1 RNA.
Input Pull Down (-) BC1
Input Pull Down (-) BC1
Input Pull Down (-) BC1
Input Pull Down (-) BC1
Input Pull Down (-) BC1
Input Pull Down (-) BC1
BC1 RNA and FMRP targeted mRNAs interact in the
absence of protein.
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Conclusions

• Translation of key mRNAs is upregulated in
• FMR1 KO mice.
• Translational repression is stronger at
  synapses.
• BC1 RNA determines specificity of mRNAs to be
• regulated by FMRP.
• FMRP and BC1 RNA associate directly.
• The association between BC1 RNA and
  FMRP-regulated mRNAs occurs in
• the absence of proteins.
• FMRPs role in translational repression at
  synapses likely underlies
• abnormalities in dedritic spine morphology in
  FRAXA patients and in FMR1
• KO mice.
• Mechanism of translation repression remains
  unkown.