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

Dendritic Spines

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

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

Spine Shape and Plasticity

Acitivity Dependent Metamorphosis of Spines

Heike Hering and Morgan Sheng (2001) Nature Reviews Neuroscience

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.

Fragile X Spine Morphology

A. Fragile X patient spines

B. Normal

Irwin, S. et al (2000) Cerebral Cortex

Heike Hering and Morgan Sheng (2001) Nature Reviews Neuroscience

Macromolecular Structure of the Synapse

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

FMRP Shuttling

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

FMRP and Synapse Pruning

FMRP Granules

Antar, L et al (2004) J. Neurosci

mGluR Activation Regulates FMRP Localization

Antar, L et al (2004) J. Neurosci

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

FMRP at Synapses

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;

(1) 5’ portion homologous to the Alu Lm(2) 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.

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).

Polysome/mRNP Distribution of mRNAs

Polysome/mRNP analysis

38%

13% 22

%

17%

Protein Levels for FMR1 KO and WT

Polysome/mRNP Distribution of FMRP Protein

FMRP co-sediments with monomeric 80S ribosomes and with mRNPs in total brain and in synaptoneurosomes.

Ribo protein (control)

mRNAs Associated with FMRP Complex

IP salt dependent

Input

wt KO wt KO wt KO wt KO

qRT-PCRHuman

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 requiredfor high-affinity binding.

Homology Between BC1 RNA and Regulated mRNAs

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.

BC1 RNA and FMRP targeted mRNAs interact in the absence of protein.

BC1 RNA Interacts with FMRP-Targeted mRNAs

Inpu

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Inpu

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Inpu

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(-) B

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Inpu

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Total brain RNA mixed with biotin labeled BC1 RNA.

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.