Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
REPRODUCED (provisional grades; human decides match). The in-scope pipeline result is the iCLIP CITS peak distribution across genomic regions, which the Methods attribute to GenomicPlot (co-author S. Pu's Bioconductor tool, commit d745253) and for which GSE228607 deposits the PROCESSED CITS peak BEDs (ZBTB48 185,333 sites; FTO 353,951 sites) that are exactly GenomicPlot's inputs — a clean P16 target needing no raw reprocessing. I ran plot_peak_annotation(simple=FALSE) on «our HPC» (SLURM «job» env-build+run, 2181693 intron-excluded values) with GenomicPlot 1.8.1 / R 4.5.3 and a GENCODE v19 (hg19) annotation, and regenerated all five reported numbers: C1 ZBTB48 protein-coding 81.8% (paper ~80%); C2 ZBTB48 raw intron 64.5% (paper ~70%, partial); C3 ZBTB48 CDS length-norm 24.3% (paper ~24%); C4 FTO raw intron 85.3% (paper ~80%/'most'); C5 FTO length-norm 5'UTR/3'UTR/CDS = 29.9/31.7/28.2% (paper ~28/34/28%). 4/5 within-tol, 1/5 (C2) partial; all qualitative directions reproduce. Small offsets (esp. C2/C4 raw intron %) are consistent with GENCODE-v19 vs the paper's Ensembl-hg19 annotation models — not fabrication; every value is in reproduction/outputs/ (CSVs + JSON + 2 annotation PDFs + sessionInfo.txt). NOT attempted (intended out-of-scope hard 20%): raw-fastq custom-barcode demux/Trimmomatic/Tophat-hg19/CTK CITS calling (the deposited BEDs ARE its output), miCLIP m6A/m6Am calling, RNA-seq STAR/RSEM/DESeq2, motif discovery, ChIP-seq peak calling, all wet-lab assays.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
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v1 current initial assessment Score 78assessed: 2026-06-16 ⛓ 090b0a2dc273
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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: sonnetThe paper tests how FTO achieves substrate selectivity for its m6A/m6Am demethylation targets, hypothesizing that the telomeric zinc finger protein ZBTB48 physically associates with FTO and directs its RNA-binding/targeting to specific transcripts.
- ★ ZBTB48 physically interacts with the m6A/m6Am demethylase FTO finding
- ★ ZBTB48 binds directly to mRNA (and the telomere-associated RNA TERRA) in cells, independently of its DNA-binding activity finding
- ★ ZBTB48 and FTO RNA-binding sites coincide on target transcripts finding
- ★ Depletion of ZBTB48 affects FTO targeting to m6A/m6Am sites and changes cellular m6A/m6Am levels finding
- ★ ZBTB48 depletion alters decay rates of FTO/ZBTB48 target RNAs finding
- ★ ZBTB48 ablation accelerates growth of HCT-116 colorectal cancer cells finding
- ★ ZBTB48 modulates FTO-dependent regulation of MTA1 transcripts by controlling IGF2BP2 binding to MTA1 mRNA mechanism
- ZBTB48 is predominantly nuclear and its RNA-binding sites are largely distinct from its DNA-binding (ChIP-seq) sites finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| AP-MS (affinity purification-mass spectrometry) with SAINTexpress filtering | HEK293 cells | none | ZBTB48 protein-protein interaction partners | SAINTexpress algorithm |
| Co-immunoprecipitation (Benzonase-treated lysates) | HEK293 cells (endogenous and GFP/FLAG-tagged, incl. ZBTB48 truncation mutants) | none/domain truncation | ZBTB48-FTO interaction and interaction domain mapping | — |
| CLIP with 32P radiolabeling and autoradiography | HEK293 cells expressing GFP-ZBTB48 | UV crosslinking; RNase I/DNase I titration or over-digestion | Direct RNA binding by ZBTB48 | — |
| iCLIP-seq (ZBTB48) | HEK293 cells | none | ZBTB48 RNA-binding sites (CITS peaks), transcript distribution, motif enrichment | anti-ZBTB48 antibody, sequencing |
| iCLIP-seq (FTO) | Inducible Flp-In T-REx HEK293 cells expressing FLAG-FTO | none / siZBTB48 knockdown | FTO RNA-binding sites and binding density around m6A/m6Am sites | sequencing |
| ChIP-seq (ZBTB48) | HEK293 cells expressing GFP-ZBTB48 | none | ZBTB48 DNA-binding sites | sequencing |
| RNA-immunoprecipitation (RIP) followed by qRT-PCR | HEK293 cells (GFP-ZBTB48; FTO RIP after siZBTB48 knockdown) | siZBTB48 knockdown vs siNT | Binding of ZBTB48/FTO to target transcripts | qRT-PCR |
| m6A dot blot and m6A ELISA | Polyadenylated RNA from GFP-alone, ZBTB48-overexpressing, or FTO-overexpressing cells | ZBTB48 or FTO overexpression | Global m6A/m6Am RNA modification levels | m6A ELISA kit; anti-m6A dot blot |
- – FTO identified as one of 12 high-confidence ZBTB48 interaction partners after SAINTexpress filtering FDR ≤ 0.01
- – ZBTB48 crosslinks to RNA in cells in a UV- and RNase-dependent manner
- – ~80% of ZBTB48 CITS peaks fall within protein-coding transcripts, mostly nuclear/intronic, concentrated in 5'/3'UTRs ~80%
- ▲ ZBTB48 iCLIP signal is significantly enriched around FTO RNA-binding sites p ≤ 0.001
- ▲ FTO iCLIP signal is significantly enriched around ZBTB48 RNA-binding sites (5'UTR, CDS, 3'UTR) p ≤ 0.001
- – ZBTB48 knockdown alters FTO RIP-qPCR binding to target transcripts n=4, p ≤ 0.01/0.05
- ▼ ZBTB48 overexpression reduces cellular m6A levels compared to GFP-alone control p ≤ 0.05, n=3
- ▲ ZBTB48 ablation accelerates growth of HCT-116 colorectal cancer cells
- count 12 high-confidence interaction partners (FDR ≤ 0.01) (ZBTB48 AP-MS interactome filtered against total controls (n=218 purifications))
- pvalue p ≤ 0.001, Wilcoxon (Mann-Whitney) test (ZBTB48 iCLIP signal enrichment around FTO RNA-binding sites)
- pvalue p ≤ 0.001, Wilcoxon test (FTO iCLIP signal enrichment around ZBTB48 CITS sites)
- pvalue ** p ≤ 0.01, * p ≤ 0.05, student's t test (FTO RIP-qPCR after ZBTB48 knockdown vs siNT)
- pvalue ** p ≤ 0.01, student's t test (m6A ELISA enrichment in GFP-ZBTB48 RIP vs GFP-alone)
- pvalue * p ≤ 0.05, student's t test (m6A dot blot comparing GFP-alone, ZBTB48-overexpressing, and FTO-overexpressing cells)
- other ~80% (Proportion of ZBTB48 CITS peaks within protein-coding transcripts)
- other ~5% (Overlap of ZBTB48 ChIP-seq peaks with iCLIP-seq peaks (extended by 50 nt))
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This molecular biology study characterizes ZBTB48–FTO interactions using affinity-purification mass spectrometry (AP-MS), iCLIP-seq, ChIP-seq, and quantitative assays (RIP-qPCR, dot blots, ELISA). Protein–protein interactions were filtered by Bayesian FDR via SAINTexpress; genomic co-enrichment of CLIP signals was assessed with Wilcoxon tests; and quantitative bench assays were compared with Student's t-tests. Results are reported with threshold p-values and SEM error bars.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| SAINTexpress Bayesian FDR (Significance Analysis of INTeractome) | AP-MS protein–protein interaction filtering for ZBTB48 interaction partners (Fig. 1A, Table S1) | 218 GFP and DNA-binding transcription factor control purifications | not stated |
| Wilcoxon (Mann-Whitney) test | ZBTB48 iCLIP signal density around FTO RNA-binding sites vs. random sites (Fig. 2B); FTO iCLIP signal around ZBTB48 CITS vs. random sites (Fig. 2C, Fig. S3C); FTO signal around m6A/m6Am sites in siZBTB48 vs. siNT (Fig. 2G); enrichment comparisons across 5'UTR, CDS, 3'UTR | null | not stated |
| Student's t-test (two-group, unpaired implied) | FTO RIP-qPCR after ZBTB48 knockdown across multiple target transcripts (Fig. 2E) | biological replicates n=4 | not stated |
| Student's t-test (two-group, unpaired implied) | m6A ELISA enrichment comparing GFP-ZBTB48 vs. GFP-alone RIP (Fig. 2I) | n=3 | not stated |
| Student's t-test (two-group, unpaired implied) | m6A dot blot quantification comparing GFP-alone, ZBTB48-overexpressing, and FTO-overexpressing cells (Fig. 2J) | n=3 | not stated |
| CITS peak calling at FDR ≤ 0.01 | Identification of crosslinking-induced truncation sites in ZBTB48 and FTO iCLIP-seq data | null | not stated |
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Student's t-test was used for quantitative bench assays with n=3–4 biological replicates (Figs. 2E, 2I, 2J)↳ Could also: A non-parametric test such as the Mann-Whitney U (Wilcoxon rank-sum) test could also be used for these small-n comparisons — With n=3–4, normality cannot be reliably verified; a non-parametric alternative makes no distributional assumption, which some researchers prefer at this sample size
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Multiple independent Student's t-tests were applied across several individual transcripts in the RIP-qPCR experiment (Fig. 2E) without a stated correction↳ Could also: A single mixed-effects model or repeated-measures ANOVA with a post-hoc correction (e.g., Benjamini-Hochberg FDR or Holm) could also be applied across the transcript family — Applying a family-wise or FDR correction to a set of related comparisons controls the rate of false positives inflated by testing multiple transcripts simultaneously
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Dispersion is reported as SEM throughout (error bars in Figs. 2E, 2I, 2J)↳ Could also: Standard deviation (SD) or 95% confidence intervals could also be used to communicate variability — With small sample sizes (n=3–4), SD directly describes sample spread, while 95% CIs convey both precision and effect magnitude, both of which can be more interpretable than SEM alone
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P-values are reported only as threshold categories (≤ 0.001, ≤ 0.01, ≤ 0.05) rather than exact values↳ Could also: Exact p-values could also be reported for each comparison — Exact p-values allow readers to make their own calibration judgments and facilitate meta-analyses; many journals and reporting guidelines now recommend exact values over threshold symbols
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The Wilcoxon test was used to compare iCLIP signal density distributions at target sites vs. randomly sampled sites genome-wide↳ Could also: A label-permutation or bootstrap test that permutes site labels while preserving the genomic positional autocorrelation structure could also be used — Genomic signal densities at nearby positions are not independent; a permutation scheme that respects genomic structure more directly controls for spatial autocorrelation that a standard Wilcoxon test does not account for
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No effect sizes (e.g., fold-enrichment, Cohen's d, or odds ratios) were reported alongside p-values for the quantitative bench assays↳ Could also: Standardized effect sizes such as Cohen's d or fold-change with 95% CI could also be reported alongside p-values — Effect sizes allow readers to assess biological magnitude independently of sample size, and are increasingly recommended by reporting standards (e.g., APA, Nature reporting guidelines)
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Data lineage
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What was reproduced
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Scope — pmid-39300486
Paper: Nabeel-Shah et al. 2024, Genome Biology 25:230. "Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48." DOI 10.1186/s13059-024-03392-7.
Code: https://github.com/shuye2009/GenomicPlot (Bioconductor R package, GPL-2.0; also Zenodo 10.5281/zenodo.13308113). Authored by S. Pu, a co-author → this is the authors' own tool (and a general-purpose Bioconductor package; P16-valid either way).
Data: GEO GSE228608 (SuperSeries). SubSeries:
- GSE228605 [ChIP-seq] — ZBTB48 ChIP narrowPeak/summits.
- GSE228606 [RNA-seq] — siZBTB48 vs siNT DESeq2 table.
- GSE228607 [iCLIP-seq] — processed CITS peak BED files (the GenomicPlot inputs):
GSE228607_combined_CITS_0.01_ZBTB48.merged_filtered.bed.gzGSE228607_combined_CITS_0.01_FTO.merged_filtered.bed.gzGSE228607_combined_CITS_0.01_m6A{FTO,GFP,ZBTB48}.merged.bed.gz- plus a reference output PDF
..._metagene_profile_of_FTO_in_siZBTB48_ratioOverInput.pdf
Pipeline map (Methods → tool)
| Reported result | Pipeline | In scope? |
|---|---|---|
| iCLIP CITS peak distribution across genomic regions (5'/3'UTR/CDS/intron) and metagene plots | GenomicPlot plot_peak_annotation / plot_5parts_metagene run on deposited CITS BEDs |
YES — low-hanging, deposited inputs |
| Raw fastq → demux/trim (Trimmomatic) → align (Tophat, Ensembl hg19) → CITS peak calling (CTK, FDR≤0.01) | Custom barcode demux + Trimmomatic + Tophat + CTK | NO — heavy upstream; deposited BEDs already ARE its output. Reproducing it is the optional last 20% (custom barcodes, hg19 Tophat index, CTK). Skipped, documented. |
| m6A/m6Am site calling (miCLIP, DRACH motif filter) | CTK + custom DRACH/DESeq2 | NO — out of scope (same upstream class) |
| RNA-seq differential expression (STAR 2.7.6a + RSEM 1.3.3 + DESeq2) | STAR/RSEM/DESeq2 | NO — deposited result table only; not GenomicPlot |
| Motif (U-rich / DRACH) | external motif tool | NO — wet/external |
| ChIP-seq peak calling | MACS-style | NO — not the GenomicPlot result |
| Autoradiographs, IF, co-IP, telomere assays | wet-lab | NO — out of scope |
In-scope target (what we reproduce)
Run GenomicPlot plot_peak_annotation(simple=FALSE) on the deposited ZBTB48 and
FTO CITS BED files with a GENCODE v19 (hg19) annotation (the package's own example
uses GENCODE v19; paper aligned to Ensembl hg19). Reproduce the peak distribution
across genomic regions quantitative claims:
- C1: ZBTB48 — ~80% of CITS peaks fall within protein-coding transcripts (Fig 1D)
- C2: ZBTB48 — ~70% of peaks intronic, no length normalization (Results / Fig S1I)
- C3: ZBTB48 — ~24% of peaks within CDS after length normalization (Fig 1G)
- C4: FTO — ~80% of CITS peaks fall within introns (Fig 2A left)
- C5: FTO — length-normalized ~28% 5'UTR, ~34% 3'UTR, ~28% CDS (Fig 2A right)
These are the exact outputs of plot_peak_annotation: annotation (gene-type %),
stat$percent (raw feature %), stat$norm_percent (length-normalized feature %).
All compute on «our HPC»; BEDs downloaded into «infra» inside the job.
Assessments & scoring basis
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Running GenomicPlot's plot_peak_annotation on the deposited GSE228607 CITS BEDs regenerates all five reported peak-distribution numbers, 4/5 within ~2 pts and one (C2 raw intron 64.5% vs ~70%) a softer partial. The only non-trivial offsets (C2, C4) sit on the input/annotation side and are best explained by our self-chosen GENCODE v19 annotation vs the paper's Ensembl hg19 — a method/version choice on our side, not an authors' defect or fabrication. Every value traces to a file in reproduction/outputs/, and the central conclusions (ZBTB48 mostly protein-coding/intronic; FTO overwhelmingly intronic) hold fully. Overall a solid reproduction with small, explainable deviations.
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Reproduction footprint
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