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
- 2026-09-19
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Deep full-text extraction
Model: sonnetThe paper tests whether ribosomal protein (RP) gene promoters, despite being highly expressed, possess a robust mechanism—specifically the sequence-specific transcription factor Rap1—that limits divergent noncoding transcription near its binding sites.
- ★ Rap1 represses divergent noncoding transcription at highly expressed RP gene promoters (e.g., IRT2 at RPL43B, iMLP1 at RPL40B) finding
- ★ The Rap1 binding site itself (not just the protein elsewhere) is required to repress divergent noncoding transcription at these promoters finding
- ★ A Rap1 motif proximal to a cryptic/divergent promoter is both necessary and sufficient to repress divergent transcription; increasing distance between the motif and the cryptic promoter abolishes repression finding
- ★ Rap1 represses divergent transcription independently of, and distinct from, previously described chromatin-based mechanisms mechanism
- ★ In the absence of Rap1, divergent transcription is elicited by the RSC chromatin remodeler mechanism
- ★ Misregulation of Rap1-repressed divergent transcripts (IRT2, iMLP1) alters expression of neighboring genes (IME1, MLP1) finding
- The Rap1 C-terminal domain contributes to repression of divergent transcription mechanism
- Auxin-inducible degron (AID) alleles of RP gene transcription factors generated as a resource for depletion studies resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Northern blot | S. cerevisiae (RAP1-AID, FHL1-AID, IFH1-AID, SFP1-AID, hmo1Δ, crf1Δ strains) | auxin-induced degron depletion (IAA) or gene deletion | IRT2, iMLP1/SUT242, SNR190 transcript levels via 32P-labeled probes | — |
| Western blot | S. cerevisiae AID strains | IAA-induced protein depletion | Protein levels of Fhl1, Ifh1, Sfp1, Rap1 (Hxk1 loading control) | — |
| RNA sequencing (poly(A) and total RNA-seq) | S. cerevisiae RAP1-AID cells | Rap1 depletion (IAA) vs DMSO control | Genome-wide sense/antisense transcript expression changes around Rap1 binding sites | — |
| Chromatin immunoprecipitation (ChIP) | S. cerevisiae RPL43B promoter mutant strains (bsΔ, bsΔS, SU, SD) | Rap1 binding site deletion/spacer insertion | Rap1 occupancy at RPL43B promoter, normalized to ACT1 | — |
| Fluorescent reporter imaging | S. cerevisiae with pPS/R1p/R1d/R1prv reporter constructs | Insertion of Rap1 motifs at proximal/distal positions; Rap1 depletion (IAA) | YFP (SUT129/noncoding) and mCherry (PPT1/coding) fluorescence signal | — |
| TSS-seq (transcription start site sequencing) | S. cerevisiae WT and RAP1-AID cells | Rap1 depletion (IAA) | Genome-wide transcription start site positions/signal near Rap1 binding sites | — |
| Single-cell transcript counting | S. cerevisiae diploid cells (WT, RPL43B-bsΔ) | Rap1 binding site deletion | IME1 transcript count per cell | — |
- ▲ Rap1 depletion (RAP1-AID + IAA) strongly induces IRT2 expression at RPL43B and multiple divergent transcripts (including iMLP1) at RPL40B, while depletion of Fhl1, Ifh1, Sfp1, or deletion of HMO1/CRF1 has no effect
- ▲ Deletion of Rap1 binding sites (RPL43B-bsΔ, RPL40B-bsΔ) increases IRT2/iMLP1 expression to levels comparable to Rap1 depletion
- ▲ Spacer insertion downstream (SD) but not upstream (SU) of the Rap1 motif relative to RPL43B derepresses IRT2, without affecting Rap1 binding by ChIP
- – A proximal Rap1 motif (R1p, 20 bp from TSS) in a reporter lowers YFP (noncoding SUT129) while increasing mCherry (coding PPT1); a distal motif (R1d, 104 bp) has little effect
- ▲ ~40% of 564 Rap1 binding sites show >2-fold increased RNA expression in 50-100 bp windows after Rap1 depletion, decreasing to 30% and 16% for 200 and 500 bp windows >2-fold
- ▲ Median IME1 transcripts per cell increases from 5 (WT) to 16 (RPL43B-bsΔ) ~3.2-fold
- ▼ Sense GUT1 expression decreases due to antisense divergent transcript from RPL8A promoter 1.7-fold
- ▲ 82% of nearest TSSs to Rap1 binding sites are antisense vs 18% sense; ~50% of promoters show >2-fold increased TSS signal within 50 bp of the Rap1 motif upon depletion >2-fold
- count 564 Rap1 binding sites (genome-wide RNA-seq window analysis around Rap1 sites)
- count 141 Rap1-regulated promoter Rap1 sites (subset of well-annotated promoters used for clustering/TSS analysis)
- count 87 Ume6-regulated promoters (control gene set unaffected by Rap1 depletion)
- fold_change >2-fold increase in RNA expression at ~40% of Rap1 sites (50-100 bp windows) (RNA-seq expression change upon Rap1 depletion)
- pvalue p < 0.0001 (IME1 transcript count comparison, WT vs RPL43B-bsΔ (unpaired Student's t test))
- mean median IME1 transcripts: 5 (WT) vs 16 (RPL43B-bsΔ) (single-cell transcript counting)
- fold_change 1.7-fold reduction in sense GUT1 expression (antisense divergent transcript from RPL8A promoter)
- other 82% antisense vs 18% sense nearest TSS to Rap1 binding site (TSS-seq distance distribution analysis)
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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