Experiment / E1VC9W3SHAAV-MPRA / in vivo MPRA

In vivo SN-MPRA single-nucleotide mutagenesis library

In Vivo Massively Parallel Reporter Assay Reveals Sequence Determinants of mRNA Localization in Astrocytes

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.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CL:0000127
Reference genome: mm10
Design focus: Variant-focused
Region of interest: chr2:102781535-102790785,chr11:55394155-55395585

Perturbation & assay details

Basal / Untreated; AAV9 single-nucleotide mutagenesis SN-MPRA library injected into P1 mouse cortex

The library contained center-190-bp consensus fragments from selected Sparc and Glt1a/Slc1a2 3′-UTR groups, all three alternate bases at each position, and 20 shuffled controls per group. Each sample was sequenced on two lanes that were merged before CPM normalization. The repository analysis uses replicate as a random effect and reports Δribosome occupancy (Ctx-TRAP/Ctx Input), Δlocalization (SN Input/Ctx Input), and Δlocal translation (PAP-TRAP/Ctx-TRAP).

Processed data

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

Column dictionary · 40 definitions

element_id: Unique element identifier from the mutagenesis results summary.
gene: Source gene or control label.
element_group: Selected 3′-UTR group or control group to which the construct belongs.
construct_class: Construct category: wild-type tile, wild-type 190-bp fragment, single-nucleotide substitution, shuffled control, or assay control.
variant: Compact variant label: reference WT, SHUFFLED, CONTROL, or source mutation such as A100C.
sequence: Reporter insert sequence, written 5′ to 3′.
sequence_length_nt: Length of the reporter insert in nucleotides.
utr_position_start: Source 3′-UTR-relative start coordinate for the construct.
utr_position_end: Source 3′-UTR-relative end coordinate for the construct.
mutation_position_in_fragment_1based: 1-based position within the 190-bp element-group fragment for a single-nucleotide substitution; blank for non-mutants.
original_nt: Source wild-type nucleotide or wt label.
mutant_nt: Source substituted nucleotide or wt label.
original_gc_percent: GC percentage of the unmutated/reference sequence as reported by the source.
predicted_deltaG_kcal_per_mol: Predicted RNA folding free energy in kcal/mol.
rG4_prediction: Source rG4-detector prediction score; blank where the source reports NA.
aav_mean_count: Mean source element count across the three AAV DNA input replicates after lane merging.
aav_mean_cpm: Mean source element CPM across the three AAV DNA input replicates after lane merging.
aav_n_replicates: Number of AAV DNA replicates with a numeric source CPM.
ctxin_mean_count: Mean source element count across retained cortex-input replicates after paper/repository outlier removal.
ctxin_mean_cpm: Mean source element CPM across retained cortex-input replicates.
ctxin_n_replicates: Number of retained cortex-input replicates with a numeric source CPM.
ctxtrap_mean_count: Mean source element count across retained cortex-TRAP replicates.
ctxtrap_mean_cpm: Mean source element CPM across retained cortex-TRAP replicates.
ctxtrap_n_replicates: Number of retained cortex-TRAP replicates with a numeric source CPM.
snin_mean_count: Mean source element count across retained synaptoneurosome-input replicates.
snin_mean_cpm: Mean source element CPM across retained synaptoneurosome-input replicates.
snin_n_replicates: Number of retained synaptoneurosome-input replicates with a numeric source CPM.
sntrap_mean_count: Mean source element count across retained PAP-TRAP/synaptoneurosome-TRAP replicates.
sntrap_mean_cpm: Mean source element CPM across retained PAP-TRAP/synaptoneurosome-TRAP replicates.
sntrap_n_replicates: Number of retained PAP-TRAP/synaptoneurosome-TRAP replicates with a numeric source CPM.
ctxin_over_aav_mean_cpm_log2_ratio: Derived log2(mean cortex-input CPM / mean AAV DNA CPM); not the paper’s mixed-model expression statistic.
ribosome_occupancy_delta_log2fc: Author-reported mutagenesis Δribosome-occupancy log2 effect estimate, normalized within element group.
ribosome_occupancy_pvalue: Author-reported p-value for the mutagenesis ribosome-occupancy comparison.
ribosome_occupancy_fdr: Author-reported Benjamini–Hochberg FDR for mutagenesis ribosome occupancy.
localization_delta_log2fc: Author-reported mutagenesis Δlocalization log2 effect estimate, normalized within element group.
localization_pvalue: Author-reported p-value for mutagenesis RNA localization.
localization_fdr: Author-reported Benjamini–Hochberg FDR for mutagenesis RNA localization.
local_translation_delta_log2fc: Author-reported mutagenesis Δlocal-translation log2 effect estimate, normalized within element group.
local_translation_pvalue: Author-reported p-value for mutagenesis local translation.
local_translation_fdr: Author-reported Benjamini–Hochberg FDR for mutagenesis local translation.

Quality control

Used the paper/repository processing: CPM normalization, lane merging, PCA-distance outlier removal of snin_10, ctxin_16/snin_16, and sntrap_3/sntrap_4, and exclusion of the low-expression Glt1a 2621–2681 group reported as not cloned well. The generated table contains 4,175 rows: 4,168 retained test constructs plus 7 assay controls. Nonsignificant constructs remain; p-values/FDRs are source results, not filters.

Curation notes

The raw S6/S8 workbook contains 4,769 element rows, including 594 constructs in slc1a2.1_2621_2681; that entire group is excluded here because the paper explicitly says it was not further analyzed. The seven control rows are retained as valid library controls but have blank inferential effect fields by design. Blank rG4 values reflect source NA values.

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