Mapping the mechanisms of protein synthesis-dependent synaptic plasticity
Neuronal HITS-CLIP. In this proposal, HITS-CLIP will be used to analyze RNA-protein interactions in the context of synaptic dynamics. In CLIP, brain tissue is UV-irradiated (step 1), covalently crosslinking in situ RNA-protein complexes in direct (~1 Ångstrom) contact. After cell lysis and partial RNase digestion (2), reducing crosslinked RNA to ~50 nt, protein-RNA complexes are purified (3-6), protein removed with proteinase K (7), RNA linkers added and RT-PCR products analyzed using high throughput sequencing methods (HITS-CLIP) (8-9). The output (right) allows organization of overlapping tags into clusters, and mapping onto the transcriptome delineates native RNA-protein interaction sites. Details can be found in recent reviews.