{
  "frozen_utc": "2026-09-19T05:13:46.437106+00:00",
  "id": "021",
  "title": "Can five retained transcribed bases introduce a reporter start-codon effect?",
  "status": "Frozen pilot: source validity and chromosome-1 test",
  "question": "Do ATG start codons within the five native bases retained after the annotated transcription start predict reduced reporter RNA enrichment beyond ordinary promoter sequence composition?",
  "rationale": "The 2021 plant core-promoter assay uses positions -165 to +5, followed by a common histone 5-prime UTR. The authors deliberately excluded longer native UTRs because they can affect RNA post-transcriptionally. A residual ATG in the retained five bases could potentially open a competing reading frame in the common reporter context. This is a hypothesis about an assay-specific sequence effect, not a claim of a new translation mechanism.",
  "minimal_test": "Use Arabidopsis chromosome 1 only, native promoters present in both assay hosts and both replicates, with at least 10 DNA-eligible barcodes per measurement at the established DNA cutoff 5. Require at least 50 last-five-base ATG carriers and at least 500 total promoters. Model log2 enrichment separately in leaf and maize protoplast, using sequence composition, GC polynomial, positional GC, a fixed TATA indicator and mono-nucleotide identity at the five retained bases. The ATG indicator tests a sequence interaction beyond these single-base terms; also report an upstream ATG count in the 30 bases before the retained segment. Test both the retained-median and pooled-count estimators. Advance only if all four ATG effects are <= -0.5 log2 units with HC3 |t| >=3. Do not inspect other chromosomes or promoter species for outcomes unless this gate passes.",
  "novelty": "After the minimal test, review upstream-ORF and reporter-assay work plus reuse of this exact dataset. Upstream translation and effects of native UTRs are already established. Source-method inspection is required to determine whether an ATG could be transcribed in the actual construct.",
  "limitations": "Annotated TSSs are not measured reporter initiation sites. A terminal-ATG association might reflect initiator sequence preferences rather than translation, and cannot establish a uORF mechanism without independent evidence. This is an intentionally stringent feasibility/effect pilot.",
  "decision": "Run the frozen pilot and retain all results; no threshold or source expansion after seeing outcomes.",
  "sources": [
    {
      "title": "Jores et al. 2021 source study",
      "url": "https://doi.org/10.1038/s41477-021-00932-y"
    },
    {
      "title": "Author-hosted full paper and assay design",
      "url": "https://queitschlab.gs.washington.edu/pdf/Jores_et_al_2021.pdf"
    }
  ],
  "searches": [
    "Jores 2021 plant core promoters upstream AUG reporter transcription start site STARR seq 170 30",
    "plant core promoter MPRA upstream open reading frame ATG confounding reporter RNA"
  ],
  "artifacts": [
    {
      "title": "Frozen protocol",
      "path": "round-003/evidence/021_protocol.json"
    }
  ]
}