Samantha Mendonsa, Nicolai von Kügelgen, Sayaka Dantsuji, Maya Ron, Laura Breimann et al.
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.
Samantha Mendonsa, Nicolai von Kügelgen, Sayaka Dantsuji, Maya Ron, Laura Breimann, Artem Baranovskii, Inga Lödige, Marieluise Kirchner, Meret Fischer, Nadja Zerna, Lucija Bujanic, Philipp Mertins, Igor Ulitsky, Marina Chekulaeva. Massively parallel identification of mRNA localization elements in primary cortical neurons. 2023-01-16. https://doi.org/10.1038/s41593-022-01243-x
A 4,813-element library of 75–110-nt tiles spanning selected mouse 3′ UTRs was cloned downstream of GFP in a pooled lentiviral reporter and assayed after neurite/soma fractionation. The packaged table contains the published baseline WT localization scores and the reported shElavl234/shScramble and depolarization comparisons.
A 6,266-sequence follow-up library derived from 16 neurite-enriched tiles tested WT controls, single-base substitutions, 2/5/10-nt transition windows, scrambled controls and motif-directed mutations/deletions. The integrated lentiviral reporter was assayed in baseline PCNs and with Ago2, Hbs1l or scrambled shRNA perturbations.
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.
Download all 4 files (ZIP)41593_2022_1243_MOESM2_ESM.xlsxE-MTAB-10902.idf.txtE-MTAB-10902.sdrf.txtREADME.txt