![]() ![]() However, mechanisms underlying how these transcripts become localized, including sequences within them required for localization, are almost completely unknown. High-throughput sequencing of RNA samples derived from these fractions have defined a fairly consistent set of transcripts that are neurite-enriched ( 19). In general, these approaches involve isolating and sequencing RNA from cells grown on microporous membranes that allow mechanical fractionation into cell body and neurite fractions ( 4, 18–22). In recent years, transcriptomic approaches have been applied to the study of RNA localization, particularly in neuronal cells. Despite the ability of MPRAs to obtain insights into gene regulation, they have not yet been widely used in the study of RNA localization. MPRAs therefore allow the rapid and unbiased detection of active regulatory elements from broad sequence pools. The abundance of each member of the MPRA library can then be quantified, and MPRA members associated with particular phenotypes or processes are identified. These sequences are integrated into reporter constructs, expressed in cells, and then populations of cells or nucleic acids are isolated based on the phenotype or process to be tested. Sequences to be tested can be random or naturally occurring elements drawn from existing genomes. These methods test thousands of potential regulatory sequences in parallel in a single experiment. Massively parallel reporter assays (MPRAs) have been used to identify regulatory mechanisms underlying a variety of gene expression regulatory processes including transcription ( 11), RNA splicing ( 12, 13), RNA stability ( 14), lncRNA nuclear localization ( 15, 16) and protein abundance ( 17). With the exception of a handful of examples ( 10), the identity of the localization regulatory sequence and the RBP that recognizes it are unknown. These sequences are often found in the 3′ UTR of the localized transcript and function through the recruitment of RNA-binding proteins (RBPs) that mediate the transport ( 9). In general, the localization of these RNAs is thought to be controlled by sequence elements, often termed ‘zipcodes’, that mark an RNA as one to be transported to a specific subcellular location ( 1, 7, 8). The localization of many of these RNAs is critical for specific cellular functions and developmental patterning ( 2, 5, 6). ![]() In a variety of cell types across a range of species, thousands of RNA molecules are asymmetrically distributed within cells ( 1–4). These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. The localization elements were enriched in adenosine and guanosine residues. 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 and human neuronal cells. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3′ UTRs. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. Hundreds of RNAs are enriched in the projections of neuronal cells.
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