HIGH-THROUGHPUT IDENTIFICATION OF RNA LOCALIZATION ELEMENTS REVEALS A REGULATORY ROLE FOR A/G RICH SEQUENCES

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequences within them that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we designed and deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous 3′ UTRs. We identified peaks of regulatory activity within several 3′ UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites. These active localization elements were rich in adenosine and guanosine residues. They were also required to be dozens of nucleotides long as subsequences showed significantly reduced activity. Using RNA affinity and mass spectrometry, we found that the RNA-binding protein Unk was associated with the active localization elements. Depletion of Unk in cells reduced the neurite-enrichment of the active localization, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization. Massively parallel reporter assay designed to identify sequences that regulate RNA transport in neuronal cells