Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor.
Part of the results reproduced; minor but material deviations remained.
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This paper has a computational component, but its primary data is legally or ethically access-restricted — identifiable patient cohorts, rare-disease genomes, or controlled-access biobanks that cannot be openly shared. The reproduction therefore could not be attempted. That is a neutral verdict: it does not mean the result is wrong or that the authors fell short — only that, for legitimate privacy reasons, it cannot be independently checked from public data. We deliberately do NOT assign a 0–100 score here, because a low number would wrongly read as a failed reproduction.
▸Reproduction agent’s raw note
DROP (no_data_accession). Control-plane screening only (zero compute). RSEM repo resolves (HTTP 200). The provided dataset pointer GSE56994 mismatches the target paper: GEO reports it as the 2014 Scc2/Scc4 cohesin SuperSeries (Series_pubmed_id=25173104), not PMID 30576656. Without a data accession matching the 2018 paper, RSEM cannot be applied to the paper's own data, so no reported value could be reproduced or compared. Not attempted: deeper text-mining for the paper's correct GEO/SRA accession, env build, any «our HPC» job — finalized per operator order. Honest outcome: the data pointer for this RU is wrong/unresolvable to this publication; flag for re-screening with a corrected accession before any reproduction attempt.
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v1 current initial assessmentassessed: 2026-06-16 ⛓ bc69238b8b62
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- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusHow is divergent noncoding transcription restricted at highly expressed gene promoters? The paper tests the hypothesis that the sequence-specific transcription factor Rap1 represses divergent noncoding transcription near its binding sites at ribosomal protein gene promoters in yeast.
- ★ Depletion of Rap1 induces divergent noncoding transcription at a large fraction of Rap1-regulated gene promoters finding
- ★ Rap1 prevents transcription initiation at cryptic promoters near its binding sites, typically within 50 bp of the Rap1 motif mechanism
- ★ Repression of divergent transcription by Rap1 is uncoupled from transcription regulation in the protein-coding direction finding
- ★ A proximal Rap1 motif is both required and sufficient to repress divergent noncoding transcription finding
- ★ Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic/divergent transcription mechanism
- ★ Divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler mechanism
- ★ Mis-regulation of Rap1-repressed divergent transcripts (IRT2, iMLP1) affects neighboring gene expression (IME1, MLP1) finding
- The Rap1 C-terminal domain contributes to repressing divergent transcription mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Northern blot | S. cerevisiae (RAP1-AID, FHL1/IFH1/SFP1-AID, hmo1Δ, crf1Δ, bs deletion/spacer mutants) | auxin-inducible degron depletion (IAA), gene deletion, Rap1 motif deletion/spacer integration | IRT2, iMLP1/SUT242, SNR190 transcript expression | 32P-labeled probes |
| Western blot | S. cerevisiae AID-tagged TF strains; Mlp1-V5 strains | IAA-induced depletion; RPL40B-bsΔ | Rap1/TF protein levels (Hxk1 loading control); Mlp1-V5 protein levels | — |
| Total and poly(A) RNA-seq | S. cerevisiae RAP1-AID (FW3877), WT (FW629) | Rap1 depletion (IAA vs DMSO) | genome-wide normalized RNA reads (Watson/Crick strands) around 564 Rap1 binding sites | — |
| TSS-seq (transcription start site sequencing) | S. cerevisiae WT (FW629) and RAP1-AID + IAA (FW3877) | Rap1 depletion | TSS positions/signal near 141 promoter Rap1 binding sites | — |
| Chromatin immunoprecipitation (ChIP) | S. cerevisiae RPL43B promoter spacer mutant strains | spacer insertion relative to Rap1 binding sites | Rap1 occupancy at RPL43B promoter, normalized over ACT1 | — |
| Single-molecule RNA FISH / single-cell transcript counting | S. cerevisiae diploid WT (FW631) and RPL43B-bsΔ (FW6139) | Rap1 binding site deletion | IME1 transcript count per cell | — |
| Fluorescent reporter imaging | S. cerevisiae with pPS divergent reporter (PPT1-mCherry / SUT129-YFP), R1p/R1d Rap1-motif constructs | Rap1 motif integration (proximal/distal); Rap1 depletion (IAA) | YFP and mCherry fluorescence signal (arbitrary units) | — |
| Rap1 domain complementation | RAP1-AID cells expressing Rap1 fragments (FL 1-827, ΔN 339-827, ΔC 1-599) | Rap1 depletion + ectopic Rap1 truncation expression | IRT2 and iMLP1 expression (northern blot) | — |
- ▲ Rap1-depleted cells show strong induction of divergent IRT2 (RPL43B) and iMLP1 (RPL40B), while depletion of Fhl1/Ifh1/Sfp1 or hmo1Δ/crf1Δ had no effect
- ▲ Deletion of Rap1 motifs (RPL43B-bsΔ, RPL40B-bsΔ) increased IRT2/iMLP1 expression to levels comparable to Rap1 depletion
- ▲ For 50/100 bp windows ~40% of Rap1 binding sites showed >2-fold increased RNA upon Rap1 depletion, decreasing to 30% (200 bp) and 16% (500 bp) ~40% at 50/100 bp; 30% at 200 bp; 16% at 500 bp
- ▲ Median IME1 expression increased from 5 transcripts/cell (WT) to 16 (RPL43B-bsΔ) 5 to 16 transcripts/cell
- ▼ Sense GUT1 expression reduced upon divergent transcription from RPL8A promoter 1.7-fold
- ▲ ~50% of promoters displayed >2-fold increased TSS signals within 50 bp of the Rap1 motif upon depletion; 82% antisense vs 18% sense nearest TSS >2-fold; 82% antisense / 18% sense
- – Integrating a 400-bp spacer downstream (SD) but not upstream (SU) of Rap1 motifs allowed IRT2 expression, while spacer did not affect Rap1 binding 400 bp spacer
- ▼ Proximal Rap1 motif (R1p) lowered SUT129-YFP while increasing PPT1-mCherry; distal motif (R1d) had little effect
- count 16 out of 138 RP gene promoters display an annotated divergent noncoding transcript (few RP promoters have annotated CUT/SUT)
- count 564 annotated Rap1 binding sites (genome-wide RNA-seq windowing analysis)
- count 141 Rap1 binding sites at well-annotated Rap1-regulated promoters; 87 Ume6-regulated control promoters (orientation/cluster and TSS analysis)
- fold_change ~40% of Rap1 sites >2-fold increased RNA (50/100 bp windows) (window-size RNA-seq analysis)
- fold_change 1.7-fold reduction of sense GUT1 expression (RPL8A divergent transcript over GUT1)
- mean median IME1: 5 (WT) vs 16 (RPL43B-bsΔ) transcripts/cell (single-cell IME1 counts; p<0.0001 unpaired Student's t test)
- pvalue p < 0.0001 (IME1 transcript count WT vs RPL43B-bsΔ, unpaired Student's t test)
- count 82% antisense, 18% sense nearest TSS to Rap1 binding site (TSS orientation near Rap1 sites)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study combines molecular assays (northern blot, western blot, ChIP, fluorescent reporter imaging, single-molecule RNA FISH) with genome-wide sequencing (total and poly(A) RNA-seq and TSS-seq) to characterize Rap1-dependent repression of divergent noncoding transcription in yeast. Most genomic results are reported descriptively through normalized read tracks, violin/box-and-whisker plots, scatterplots, and k-means-clustered heatmaps over defined windows around Rap1 binding sites, while a single explicit hypothesis test (an unpaired Student's t test for single-cell IME1 transcript counts) is reported. Quantitative summaries are shown as mean ± SEM (ChIP) or mean + 95% confidence intervals (reporter imaging) with the underlying n stated per panel.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| unpaired (two-sample) Student's t test | IME1 transcripts per cell, WT vs RPL43B-bsΔ (Figure 1G) | n = 139 cells | not stated |
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Single-cell IME1 transcript counts were compared with an unpaired Student's t test (Figure 1G).↳ Could also: A nonparametric Mann-Whitney U test (or a count-based model such as negative binomial/Poisson regression) could also be applied. — Single-molecule transcript counts are discrete and often right-skewed; a rank-based or count model makes fewer normality assumptions and can be preferred when comparing medians, which the figure already reports.
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ChIP data were summarized as mean ± SEM with n = 3 (Figure 3C).↳ Could also: Showing SD or a 95% confidence interval, and overlaying the individual replicate points, could also be used. — For small n, SD or a CI conveys the spread and uncertainty more directly than SEM, and plotting individual replicates makes the underlying data fully visible.
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Genome-wide expression changes across windows around Rap1 sites were summarized descriptively (fold-change thresholds, violin/box plots) without an associated test (Figures 2C–2H).↳ Could also: A differential-expression framework with multiple-testing control (e.g., DESeq2/edgeR or limma with Benjamini-Hochberg FDR) could also quantify these changes. — A modeled approach with FDR control assigns calibrated significance to per-site or per-window changes across the genome and complements the descriptive fold-change cutoffs.
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Promoters were grouped using k-means clustering with k = 3 (Figure 2F).↳ Could also: Reporting a cluster-number selection criterion (e.g., silhouette, gap statistic) or comparing with hierarchical clustering could also be presented. — An explicit selection metric documents how k was chosen and conveys the stability of the chosen clusters.
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Reporter imaging values were reported as mean + 95% CI with n = 50 cells per sample (Figure 3E).↳ Could also: A formal group comparison (e.g., ANOVA with a post-hoc correction, or a mixed model accounting for replicate/experiment) could also accompany the CIs. — An accompanying test with multiplicity control would quantify the differences among the multiple reporter conditions while controlling the family-wise error rate.
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Significance for the t test was reported as a threshold (∗p < 0.0001) (Figure 1G).↳ Could also: Reporting the exact p value alongside an effect-size estimate (e.g., difference in medians/means with its CI) could also be done. — Exact p values and effect sizes give readers the magnitude and precision of the effect rather than only whether a threshold was crossed.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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IME1 transcript levels increase from a median of 5 to 16 transcripts per cell in RPL43B Rap1-binding-site deletion cells as measured by single-molecule RNA FISH.imaging s-cerevisiae up 2018×1papers★ This paper is the founder (earliest)
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Integration of a proximal Rap1 motif represses divergent SUT129 noncoding reporter expression, while a distal motif has little effect.imaging s-cerevisiae down 2018×1papers★ This paper is the founder (earliest)
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TSS-seq reveals greater than 2-fold increased divergent TSS signals at approximately 50% of Rap1-associated promoters upon Rap1 depletion, with 82% of new TSSs oriented antisense.other s-cerevisiae up 2018×1papers★ This paper is the founder (earliest)
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A 400-bp spacer inserted downstream but not upstream of the Rap1 binding motif derepresses IRT2 transcription without displacing Rap1 from chromatin, indicating a positional requirement for repression.other s-cerevisiae mixed 2018×1papers★ This paper is the founder (earliest)
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IRT2 and iMLP1 divergent noncoding RNAs are upregulated upon Rap1 depletion in S. cerevisiae; depletion of co-factors Fhl1, Ifh1, Sfp1, Hmo1, or Crf1 has no effect.other s-cerevisiae up 2018×1papers★ This paper is the founder (earliest)
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Genome-wide RNA-seq shows approximately 40% of Rap1 binding sites have greater than 2-fold increased divergent noncoding RNA upon Rap1 depletion.RNA-seq s-cerevisiae up 2018×1papers★ This paper is the founder (earliest)
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Sense GUT1 mRNA expression is reduced 1.7-fold when divergent transcription is induced from the RPL8A promoter.RNA-seq s-cerevisiae down 2018×1papers★ This paper is the founder (earliest)
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This RU was a drop (no_data_accession) with zero compute: the supplied accession GSE56994 resolves to an unrelated 2014 cohesin SuperSeries (Series_pubmed_id=25173104), not the 2018 divergent-noncoding-transcription paper (PMID 30576656). The blocker is on the data-availability / screening side (a wrong data pointer, and the paper's true accession was admittedly not exhaustively mined), not an authors' defect or a fabrication signal. Consequently q1/q2 are red (no comparable input or endpoint), while q5/q7/q8 stay yellow — derivability and the core claim could not be assessed because no reproduction was attempted, so flagging them critical-red would overstate the evidence. Recommend re-screening with a corrected GEO/SRA accession before any reproduction.
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