{
  "frozen_utc": "2026-09-19T05:19:03.610327+00:00",
  "id": "022",
  "title": "Do cryptic splice-donor motifs predict post-transcriptional loss in intronic reporters?",
  "status": "Frozen minimal test",
  "question": "In a plant MPRA where all fragments are placed inside a reporter intron, do strong sense-strand splice-donor motifs predict lower total-versus-newly-labeled RNA abundance than reverse-complement motifs?",
  "rationale": "Voichek et al. 2024 measured both total mRNA and mRNA labeled during a 20-minute 5-EU pulse for the same approximately 12,000 reporter fragments. Correct splicing removes each variable insert, making fragment-dependent post-transcriptional differences an interesting possibility. An internal GT[AG]AGT donor could compete with the canonical donor; its reverse-complement sequence is a specificity control. Splicing effects are established biology, and the proposed assay mechanism requires independent support.",
  "minimal_test": "Use only Arabidopsis chromosome-1 origin fragments from Supplementary Table 5, including all three native positions, with finite paired total/5-EU values and exactly 160 A/C/G/T bases. Primary outcome is the published total-RNA minus newly-labeled-RNA log2 value. Regress on sense donor count GT[AG]AGT and reverse-complement count ACT[CT]AC, mono/dinucleotide composition, GC squared/cubed, fixed origin-position categories, and the known YVGATCBR regulatory motif count. Report the sense-minus-antisense coefficient contrast with standard errors clustered by source gene; a gene-cluster bootstrap will be used in validation if needed. Require >=500 fragments, >=50 sense-motif carriers and >=50 reverse-motif carriers; advance only for contrast <=-0.30 log2 and t<=-3. Other chromosomes and other MPRA outcomes remain unexamined until this gate passes.",
  "novelty": "After a passing pilot, search for cryptic-splicing effects in plant reporter libraries, previous reanalysis of this dataset and transcriptome evidence for insert retention. Known intron-mediated transcription enhancement and GATC effects are not new.",
  "limitations": "A total/newly-labeled ratio is not a calibrated RNA half-life. The short labeling pulse, capture efficiency and transient expression may matter. A motif association does not prove altered splicing; follow-up RNA sequencing would be needed to support that mechanism.",
  "decision": "Run the frozen paired-RNA pilot; do not change motif, threshold or sampling scope after outcomes.",
  "sources": [
    {
      "title": "Voichek et al. 2024, Nature Genetics",
      "url": "https://doi.org/10.1038/s41588-024-01907-3"
    },
    {
      "title": "Published supplementary data",
      "url": "https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41588-024-01907-3/MediaObjects/41588_2024_1907_MOESM4_ESM.xlsx"
    }
  ],
  "searches": [
    "\"Widespread position-dependent transcriptional regulatory sequences in plants\" data github",
    "\"Widespread position-dependent\" splicing motifs intron"
  ],
  "artifacts": [
    {
      "title": "Frozen protocol",
      "path": "round-003/evidence/022_protocol.json"
    },
    {
      "title": "Pinned source hashes",
      "path": "round-003/evidence/022_source_downloads.json"
    }
  ]
}