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

2. Dendritic Spines
Nimchinsky, E. et al (2002) Annual Review of Physiology

3. Spine Shape and Plasticity
Nimchinsky, E. et al (2002) Annual Review of Physiology

4. Acitivity Dependent Metamorphosis of Spines
Heike Hering and Morgan Sheng (2001) Nature Reviews Neuroscience

5. 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.

6. Fragile X Spine Morphology
Fragile X patient spines
Normal
Irwin, S. et al (2000) Cerebral Cortex

7. Macromolecular Structure of the Synapse
Heike Hering and Morgan Sheng (2001) Nature Reviews Neuroscience

8. FMRP Shuttling
Claudia Bagni and William T. Greenough (2005) Nature Reviews Neuroscience

9. FMRP and Synapse Pruning
Claudia Bagni and William T. Greenough (2005) Nature Reviews Neuroscience

10. FMRP Granules
Antar, L et al (2004) J. Neurosci

11. 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

13. 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.

15. 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).

16. Polysome/mRNP Distribution of mRNAs
22%
13%
17%
38%
Polysome/mRNP analysis

17. Protein Levels for FMR1 KO and WT

18. 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.

19. mRNAs Associated with FMRP Complex
IP salt dependent
Human
qRT-PCR
Input wt KO wt KO wt KO wt KO

20. 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.

21. Homology Between BC1 RNA and Regulated mRNAs

22. 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.

23. BC1 RNA Interacts with FMRP-Targeted mRNAs
Total brain RNA mixed with biotin labeled BC1 RNA.
Pull Down
Input
(-) BC1
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Input
(-) BC1
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Input
(-) BC1
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Input
(-) BC1
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Input
(-) BC1
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Input
(-) BC1
BC1 RNA and FMRP targeted mRNAs interact in the absence of protein.

24. 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.
FMRP’s 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.