Martin Mikl, Davide Eletto, Malak Nijim, Minkyoung Lee, Atefeh Lafzi et al.
Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested ∼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts.
Martin Mikl, Davide Eletto, Malak Nijim, Minkyoung Lee, Atefeh Lafzi, Farah Mhamedi, Orit David, Simona Baghai Sain, Kristina Handler, Andreas E. Moor. A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons. 2022-09-26. https://doi.org/10.1093/nar/gkac806
A plasmid reporter library containing 150-nt native mouse 3′UTR tiles and designed motif or synthetic-sequence variants was transfected into differentiated CAD cells grown on 3-µm transwell inserts. Soma and neurite fractions were collected after transfection and profiled in three biological replicates each using UMI-based targeted reporter sequencing.
The same plasmid reporter library of 150-nt native mouse 3′UTR tiles and designed motif or synthetic-sequence variants was transfected into differentiated Neuro-2a cells grown on 3-µm transwell inserts. Soma and neurite fractions were collected after transfection and profiled in three biological replicates each using UMI-based targeted reporter sequencing.
Wild-type 150-nt native 3′UTR tiles from the reporter library were transfected into differentiated CAD cells in six-well plates. Transcription was inhibited with 20 ng/ml Actinomycin D and reporter RNA was measured at 0, 4, and 24 hours in duplicate samples; the table contains the published relative RNA-abundance effects.
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 17 files (ZIP)GSE173098_RNAloc_MPRA_counts.csv.gzREADME.txtsupplementary_tables/TableS1.csvsupplementary_tables/TableS10.csvsupplementary_tables/TableS11.csvsupplementary_tables/TableS12.xlsxsupplementary_tables/TableS13.xlsxsupplementary_tables/TableS14.xlsxsupplementary_tables/TableS18.txtsupplementary_tables/TableS2.csvsupplementary_tables/TableS3.csvsupplementary_tables/TableS4.csvsupplementary_tables/TableS5.xlsxsupplementary_tables/TableS6.csvsupplementary_tables/TableS7.csvsupplementary_tables/TableS8.csvsupplementary_tables/TableS9.csv