Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X
1Howard Hughes Medical Institute, Department of Human Genetics, Department of Pediatrics, Atlanta, GA 30322, USA.
Abstract:
Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
Insights
Fragile X syndrome stems from lacking FMRP protein. This study reveals FMRP binds specific mRNAs, and its absence disrupts translation, potentially causing the syndrome.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Fragile X syndrome is caused by the absence of the Fragile X mental retardation protein (FMRP).
- FMRP is an RNA-binding protein crucial for neuronal development and function.
Purpose of the Study:
- To identify messenger RNAs (mRNAs) associated with the FMRP ribonucleoprotein complex.
- To investigate the role of FMRP in mRNA translation and polyribosome profiles in fragile X syndrome.
Main Methods:
- Coimmunoprecipitation of FMRP-bound mRNAs from mouse brain followed by microarray analysis.
- Analysis of mRNA polyribosome profiles in fragile X cells using microarrays.
- Quantitative RT-PCR validation of identified mRNAs.
- In vitro assessment of FMRP interaction with G-quartet structures.
Main Results:
- Identified 432 mRNAs associated with the FMRP complex in mouse brain.
- Found 251 mRNAs with abnormal polyribosome profiles in fragile X cells, despite normal cytoplasmic levels.
- Over 50% of coimmunoprecipitated mRNAs with human orthologs were among those with altered polyribosome profiles.
- Nearly 70% of transcripts identified in both studies contained G-quartet structures, suggesting direct FMRP targeting.
Conclusions:
- Translational dysregulation of FMRP-associated mRNAs is likely the primary cause of fragile X syndrome.
- Identified candidate genes involved in the fragile X phenotype.
- G-quartet structures are important targets for FMRP-mediated translational control.
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