RNA-binding proteins are key players in post-transcriptional regulation of gene expression by orchestrating RNA fate from synthesis to decay. Hundreds of proteins with RNA-binding capacity have been identified so far, yet only a small fraction has been functionally characterized and presumably many more RNA-binding proteins await discovery. The roles of RNA-binding proteins in the nervous system are of particular interest because accumulative evidence has linked RNA-based mechanisms to neural development, maintenance and repair. Here, the three RNA-binding proteins under study are IGF-II mRNA binding proteins IMP-1 and IMP-2, known to be involved in mRNA localization, translational control and stability, and adenomatous polyposis coli (APC), identified as a novel RNA-binding protein. To systematically identify their RNA binding profiles, a high-throughput approach combining protein-RNA crosslinking and immunoprecipitation with next-generation sequencing (HITS-CLIP) was applied in embryonic mouse brain. A nonparametric method was developed to computationally analyze the CLIP sequencing data, mapping transcriptome-wide protein-RNA interactions. The identified target mRNAs of IMP-1 and IMP-2 were highly enriched for functions related to neural development, especially neuron projection morphogenesis and axon guidance signaling. Moreover, these target mRNAs were associated with a variety of neurological diseases, including neurodevelopmental and neurodegenerative disorders. Supporting roles in axon development, knockdown of IMP-1 or IMP-2 caused aberrant trajectories of commissural axons in chicken spinal cord. APC mRNA targets were highly enriched for APC-related functions, including microtubule organization, cell and axon motility, Wnt signaling, cancer and neurological disease. Among the APC targets was Tubulin β-2B (Tubb2b), previously known to be required for neuronal migration. It was found that Tubb2b was synthesized in axons, and localized preferentially to dynamic microtubules in the peripheral domain of the growth cone. Blocking the APC binding site in the Tubb2b mRNA 3'UTR caused reduction in its expression in axons and loss of the growth cone peripheral area, and impaired cortical neuron migration in vivo. These findings offer an informative snapshot of the protein-RNA interactome, which can provide a basis to better understand the roles of RNA-binding proteins in the nervous system.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11744447 |
| Date | January 2013 |
| Creators | Quan, Jie |
| Contributors | Flanagan, John Gerard |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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