Thus, we characterized global RNA editing in the zebrafish transcriptome and used a microfluidic-based high-throughput technique to accurately quantify RNA editing levels. Biochemical assays showed that Fmrp interacts with the Adar2a protein, which is increased in fmr1-/- larvae. Furthermore, similar to human patients, the fmr1-/- larvae were hyperactive. We found that loss of Fmrp altered neuronal circuit formation. Here, we used the fmr1 mutant zebrafish ( fmr1-/-), which enables high-throughput genetics and live imaging experiments in a transparent and evolutionarily conserved brain. However, it is unclear by which mechanism the loss of Fmrp affects the sequence of neuronal genes and, ultimately, brain function. This protein inhibits the production of various proteins in the brain and interacts with the Adar enzyme, which converts the nucleotide A into I in RNAs. The genetic cause of this disorder is the silencing of the fmr1 gene, which encodes the RNA-binding protein Fmrp. The most frequent inherited mental retardation disorder is fragile X syndrome, which is characterized by learning disabilities, cognitive impairment, anxiety, and hyperactive behavior. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. Utilizing the fmr1 zebrafish mutant ( fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. The RNA-binding protein Fmrp represses protein translation, particularly in synapses. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 ( fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation.
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