Experiment / E2W75NCEVSort-Seq / Flow-Seq MPRA

FACS-uORF protein-expression activity in yeast

Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning

An episomal dual-fluorescence reporter MPRA compared natural yeast transcript leaders containing individual uORFs with matched AAG start-codon mutants. YFP/mCherry fluorescence was measured by sorting cells into nine FACS bins, and targeted sequencing of each bin was used to estimate protein-expression effects in BY4741 wildtype and upf1Δ yeast.

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Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.

Organism: Budding yeast
Taxonomy ID: NCBITaxon:4932
Biosample: UNMAPPED:Saccharomyces_cerevisiae_BY4741
Reference genome: Not reported / not applicable
Design focus: Other
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated; matched BY4741 wildtype and upf1Δ strains

The ENO2 promoter drives a pGM-ENO2-YFP-mCherry reporter containing a natural 5′ transcript leader upstream of YFP; the paired mutant changes the tested uORF start codon to AAG. Cells were grown in uracil-dropout medium at 30°C to log phase, sorted into nine YFP/mCherry bins, and bin-specific reporter sequences were counted by paired-end Illumina sequencing. The processed table combines the paper's Supplementary file 1a wildtype results and 1e upf1Δ results; log2 activity is log2(wildtype reporter / AAG mutant reporter), so negative values indicate repression by the tested uORF.

Processed data

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Visible columns (67 of 67)

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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 · 67 definitions

element_id: Unique reporter-context identifier formed from the source uORF ID, source UTR ID, and assay condition.
uorf_id: Source identifier for the assayed upstream or overlapping open reading frame.
utr_id: Source identifier for the natural transcript leader containing the tested uORF.
assay_condition: Reporter host condition: wildtype_BY4741 or upf1_delta_BY4741.
source_species: Species from which the natural transcript-leader sequence was derived.
orf_type: Source annotation: uORF or overlapping ORF (oORF).
spealman_2018: Whether the source table marks the element as present in Spealman et al. 2018.
start_conservation: PhastCons-derived conservation score for the uORF start codon, when available.
uorf_conservation: PhastCons-derived conservation score for the uORF coding region, when available.
stop_conservation: PhastCons-derived conservation score for the uORF stop codon, when available.
uorf_length_nt: Length of the uORF coding sequence in nucleotides; blank for annotated overlapping ORFs without a defined stop in the source table.
cap_distance_nt: Distance from the designed transcript start/5′ cap to the uORF start codon in nucleotides.
cds_distance_nt: Distance from the uORF stop codon to the downstream YFP/main ORF start in nucleotides.
kozak_context: Sequence context surrounding the tested uORF start codon.
start_codon: Native uORF initiation codon in the reporter.
uorf_cds: Source uORF coding sequence.
uorf_cai: Codon Adaptation Index of the uORF coding sequence, when available.
uorf_peptide: Predicted peptide translated from the uORF, when available.
stop_codon: Native uORF stop codon, when available.
stop_downstream_sequence: Source sequence downstream of the uORF stop codon.
mutant_start_codon: Start codon in the matched nonfunctional reporter mutant; AAG for this assay.
mutant_construct_id: Source construct identifier for the AAG mutant reporter.
wildtype_construct_id: Source construct identifier for the native/wildtype reporter.
replicate_1_mutant_dna_rpm: Normalized plasmid-DNA read representation for the AAG mutant in FACS replicate 1.
replicate_1_wildtype_dna_rpm: Normalized plasmid-DNA read representation for the native reporter in FACS replicate 1.
replicate_1_mutant_rna_rpm: Normalized reporter-RNA read representation for the AAG mutant in FACS replicate 1.
replicate_1_wildtype_rna_rpm: Normalized reporter-RNA read representation for the native reporter in FACS replicate 1.
replicate_1_mutant_rna_level: Relative reporter RNA level for the AAG mutant in FACS replicate 1.
replicate_1_wildtype_rna_level: Relative reporter RNA level for the native reporter in FACS replicate 1.
replicate_1_mutant_yfp: FACS-derived YFP/mCherry estimate for the AAG mutant in replicate 1.
replicate_1_wildtype_yfp: FACS-derived YFP/mCherry estimate for the native reporter in replicate 1.
replicate_1_log2_wt_over_aag: Per-replicate log2(native reporter / AAG mutant) protein-expression effect in FACS replicate 1.
replicate_1_raw_p: Unadjusted Wilcoxon rank-test p-value for the native-versus-AAG comparison in replicate 1.
replicate_1_adjusted_p: Benjamini-Hochberg-adjusted p-value for the native-versus-AAG comparison in replicate 1.
replicate_2_mutant_dna_rpm: Normalized plasmid-DNA read representation for the AAG mutant in FACS replicate 2.
replicate_2_wildtype_dna_rpm: Normalized plasmid-DNA read representation for the native reporter in FACS replicate 2.
replicate_2_mutant_rna_rpm: Normalized reporter-RNA read representation for the AAG mutant in FACS replicate 2.
replicate_2_wildtype_rna_rpm: Normalized reporter-RNA read representation for the native reporter in FACS replicate 2.
replicate_2_mutant_rna_level: Relative reporter RNA level for the AAG mutant in FACS replicate 2.
replicate_2_wildtype_rna_level: Relative reporter RNA level for the native reporter in FACS replicate 2.
replicate_2_mutant_yfp: FACS-derived YFP/mCherry estimate for the AAG mutant in replicate 2.
replicate_2_wildtype_yfp: FACS-derived YFP/mCherry estimate for the native reporter in replicate 2.
replicate_2_log2_wt_over_aag: Per-replicate log2(native reporter / AAG mutant) protein-expression effect in FACS replicate 2.
replicate_2_raw_p: Unadjusted Wilcoxon rank-test p-value for the native-versus-AAG comparison in replicate 2.
replicate_2_adjusted_p: Benjamini-Hochberg-adjusted p-value for the native-versus-AAG comparison in replicate 2.
replicate_3_mutant_dna_rpm: Normalized plasmid-DNA read representation for the AAG mutant in FACS replicate 3.
replicate_3_wildtype_dna_rpm: Normalized plasmid-DNA read representation for the native reporter in FACS replicate 3.
replicate_3_mutant_rna_rpm: Normalized reporter-RNA read representation for the AAG mutant in FACS replicate 3.
replicate_3_wildtype_rna_rpm: Normalized reporter-RNA read representation for the native reporter in FACS replicate 3.
replicate_3_mutant_rna_level: Relative reporter RNA level for the AAG mutant in FACS replicate 3.
replicate_3_wildtype_rna_level: Relative reporter RNA level for the native reporter in FACS replicate 3.
replicate_3_mutant_yfp: FACS-derived YFP/mCherry estimate for the AAG mutant in replicate 3.
replicate_3_wildtype_yfp: FACS-derived YFP/mCherry estimate for the native reporter in replicate 3.
replicate_3_log2_wt_over_aag: Per-replicate log2(native reporter / AAG mutant) protein-expression effect in FACS replicate 3.
replicate_3_raw_p: Unadjusted Wilcoxon rank-test p-value for the native-versus-AAG comparison in replicate 3.
replicate_3_adjusted_p: Benjamini-Hochberg-adjusted p-value for the native-versus-AAG comparison in replicate 3.
mean_log2_wt_over_aag: Arithmetic mean of the three per-replicate log2(native/AAG) protein-expression effects.
mean_mutant_yfp: Arithmetic mean of the three AAG-mutant YFP/mCherry estimates.
mean_wildtype_yfp: Arithmetic mean of the three native-reporter YFP/mCherry estimates.
mutant_yfp_replicate_sd: Sample standard deviation of the three AAG-mutant YFP/mCherry estimates.
wildtype_yfp_replicate_sd: Sample standard deviation of the three native-reporter YFP/mCherry estimates.
mean_mutant_rna_level: Arithmetic mean of the three relative reporter RNA levels for the AAG mutant.
mean_wildtype_rna_level: Arithmetic mean of the three relative reporter RNA levels for the native reporter.
consistent_direction: Source flag indicating whether replicate effects have the same direction.
significant_fdr_0_05: Source flag indicating significance after FDR correction at 0.05.
source_multi_context: Source workbook context flag for reporters associated with multiple transcript contexts.
source_qc_pass: Whether the row passed the source-table QC and package-level validity filtering; all retained rows are true.

Quality control

The paper compared three FACS-uORF replicates and removed transcript leaders with noisy YFP measurements (standard deviation >0.05) or fewer than 50 normalized reads per uORF. Wilcoxon rank tests were performed per replicate with Benjamini-Hochberg FDR control at 5%; the paper called a regulator significant only when FDR <0.05 and the direction was consistent across replicates. Supplementary file 1a/1e are post-QC result tables. This package additionally removed one blank artifact row and retained non-significant rows with valid effect estimates.

Curation notes

The assay host is S. cerevisiae BY4741, while the reporter library includes natural transcript leaders from S. cerevisiae and S. paradoxus. The source workbook does not specify a genome assembly, so reference_genome is null. Five uORF IDs occur in multiple UTR contexts and were retained because transcript-leader position is a biological variable in this study. One source row (chrI:114243-oORF) has the literal UTR value 'Table'; it was retained verbatim as a source annotation anomaly. The processed table contains 2,038 nonblank wildtype contexts from source section 1a and 1,361 nonblank upf1Δ contexts from section 1e, including rows that were not significant regulators.

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