Ankita Arora, Roberto Castro-Gutierrez, Charlie Moffatt, Davide Eletto, Raquel Becker et al.
Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. 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. 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. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. 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. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.
Ankita Arora, Roberto Castro-Gutierrez, Charlie Moffatt, Davide Eletto, Raquel Becker, Maya Brown, Andreas E. Moor, Holger A. Russ, J. Matthew Taliaferro. High-throughput identification of RNA localization elements in neuronal cells. 2022-09-15. https://doi.org/10.1093/nar/gkac763
A densely tiled library of 260-nt mouse 3′-UTR fragments and controls was inserted into the 3′ UTR of a firefly luciferase reporter and integrated into CAD cells by Cre/loxP-mediated recombination. Targeted UMI RNA-seq of cell-body and neurite fractions, with four biological replicates per compartment, quantified each oligo's localization.
A densely tiled library of 260-nt mouse 3′-UTR fragments and controls was inserted into the 3′ UTR of a GFP reporter and integrated into Neuro-2a cells by Cre/loxP-mediated recombination. Targeted UMI RNA-seq of cell-body and neurite fractions, with four biological replicates per compartment, quantified each oligo's localization.
A densely tiled library of 260-nt mouse 3′-UTR fragments and controls was inserted into the 3′ UTR of a firefly luciferase reporter and integrated into Neuro-2a cells by Cre/loxP-mediated recombination. Targeted UMI RNA-seq of cell-body and neurite fractions, with four biological replicates per compartment, quantified each oligo's localization.
A densely tiled library of 260-nt mouse 3′-UTR fragments and controls was inserted into the 3′ UTR of a GFP reporter and integrated into CAD cells by Cre/loxP-mediated recombination. Targeted UMI RNA-seq of cell-body and neurite fractions, with four biological replicates per compartment, quantified each oligo's localization.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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