Sarah K. Koester, Kristina Sakers, Gareth M. Rurak, Stephen P. Plassmeyer, Kelli McFarland White et al.
RNA localization and local translation are essential mechanisms to fine-tune spatiotemporal gene expression in the nervous system. However, efficiently assessing the thousands of possible sequence determinants of RNA localization is a challenge, particularly for cell types that only reach morphological maturity in vivo. Here, we developed an in vivo Massively Parallel Reporter Assay (MPRA), termed Synaptoneurosomal (SN)-MPRA to enable identification of sequence determinants of mRNA localization and local translation, and applied this to astrocytes. We evaluated multiple models of RNA localization for two locally translated astrocyte mRNAs, Glt1 and Sparc, including increased transcript abundance, “zipcode” elements, and RNA secondary structure. Our results establish a high-throughput in vivo framework for identifying cis-regulatory sequences driving RNA localization and local translation, and suggest astrocytes use diverse mechanisms to regulate subcellular gene expression. More broadly, SN-MPRA offers a versatile platform to study RNA localization in vivo, where biological context and intercellular interactions are preserved.
Sarah K. Koester, Kristina Sakers, Gareth M. Rurak, Stephen P. Plassmeyer, Kelli McFarland White, Sebastian Alves Ferreira Dias, Peter Baird, Dina J. Kornbluth, Joseph D. Dougherty. In Vivo Massively Parallel Reporter Assay Reveals Sequence Determinants of mRNA Localization in Astrocytes. 2026-04-30. https://doi.org/10.64898/2026.04.27.721172
A focused in vivo AAV9 SN-MPRA tiled 130-nt fragments across astrocyte mRNA 3′ UTRs from Slc1a2/Glt1 isoforms, Sparc, and Hsbp1, with shuffled and positive controls. Reporter RNA was measured in cortex input, cortex TRAP, synaptoneurosome input, and PAP-TRAP fractions relative to AAV DNA.
A follow-up in vivo AAV9 SN-MPRA tested every possible single-base substitution across the center 190 bp of eight previously localized 3′-UTR element groups, alongside wild-type tiles, 190-bp references, shuffled controls, and assay controls. Mutagenesis effects were measured in cortex input, cortex TRAP, synaptoneurosome input, and PAP-TRAP fractions after within-group CPM normalization.
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 12 files (ZIP)analysis_code_README.mdGSE330741-GPL34281_series_matrix.txt.gzGSE330741-GPL34290_series_matrix.txt.gzGSE330741_astrocyte_SNMPRA_count_members.txtGSE330741_astrocyte_SNMPRA_counts.tarGSE330741_family.soft.gzlocalization_mpra_functions.RSN_MPRA_followup_manuscript_code.RmdSN_MPRA_manuscript_code.Rmdsource_manifest.txtsupplementary_figures.pdfsupplementary_media-1.xlsx