Massively parallel identification of mRNA localization elements in primary cortical neurons

Abstract

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called ‘zipcodes’. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3′ untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons—cell bodies and neurites—with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes.

Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of developing neurons of the cerebral cortex

“…Therefore, we profiled the location of CPEs in 3 ’UTRs of GC transcripts to investigate potential mechanisms by which CPEBs might regulate transcripts in GCs. The presence of a CPE spaced 6-25 nt from the CPSF hexamer binding site at the 3 ’ end of a transcript enables translational activation 92,93 , and the presence of a CPE in the 3 ’UTRs of several mRNAs has been associated with localization to neurites 94,95 .…”

“…Several known levels of regulation are likely to contribute to this striking result. These include long-range directed transport [38][39][40][41][42][43][44][45][46][47][48][49] and RNA stability 51,52 (reviewed in 53 ), which influence both the rate at which RNAs arrive at GCs and the duration of time they are available within the GC-localized transcriptome, respectively. We identify multiple characteristics of GC-localized RNAs that likely contribute to subcellular localization and translational regulation in the context of developmental dynamics.…”

“…Neurons are extreme examples of polarized cells, with highly specialized subcellular compartments [3][4][5][6][7][8] . The spatial enrichment of specific transcripts to distal compartments, such as axons and dendrites, is likely linked to targeted RNA trafficking based on a combination of specific motifs [9][10][11][12][13][14][15] and differential expression of RNA binding proteins (RBPs) [16][17][18][19][20] , reviewed in 22 , as well as adjustments of RNA stability and local turnover mediated by RBPs and miRNAs 23,24 , reviewed in 25 . During development, axonal GCs navigate complex extracellular environments by responding to diverse substrate-bound and diffusible guidance cues.…”

Structural determinants of co-translational protein complex assembly

“…Considering heteromers, mRNA co-localization is a prerequisite for co-translational assembly. Such co-localization could be achieved actively through zipcode sequences 63 or through partitioning into specific condensates 64 . Alternatively, random diffusion could suffice to promote nascent chain recognition 65 .…”

Spatial visualization of A-to-I Editing in cells using Endonuclease V Immunostaining Assay (EndoVIA)

“…28 Beyond Alu repeats, the 3’ UTRs of RNAs also contain cis -regulatory elements, often referred to as “zipcodes,” that influence the localization of mRNA. 30,31 These sequences serve as encoded cellular addresses that RNA-binding proteins (RBPs) interact with to facilitate trafficking. 32 Considering that A-to-I editing primarily occurs within these non-coding regions, mRNA localization is subject to change based on the extent of editing.…”