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 sequence elements 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 mouse 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 in mouse cells as well as active in human neurons. The active localization elements were enriched in adenosine and guanosine residues and were tens to hundreds of nucleotides long as shortening of the elements led to 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 elements, 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.