N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in y
Provisional — an automated or curator check raised a specific concern and points reviewers here. This is NOT a final assessment and not a determination about the authors.
The main results reproduced, with only marginal, non-material deviations.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- 🟡The central claim did not (fully) hold under reproduction
- 🟡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
PARTIAL, well-evidenced. Layer A (deposited GSE193291 binding-site BEDs vs paper) reproduces the headline CLIP counts ~1:1 -- Gis2=3563, m6A=1286, Pho92 Ime4-dep=642 (all exact), Pho92 total=3813 vs reported 3823 (within 0.26%, and 3823 is a likely typo since the paper's own 642+3171=3813). B1 peak->gene counts reproduce WITHIN-TOL (Pho92 Ime4-dep 504 vs 507; m6A 853-893 bracketing 870) -- but ONLY under an UNSTRANDED + ~300nt 3'UTR-extended assignment; strand-specific ORF-only under-counts. B2 stop-codon positional fractions (23%/13%) do NOT reproduce (1.7-7.9%) -- needs the authors' transcript-relative metagene script, which is not in the deposited read-mapper repo. B4 RNA-seq DE counts (1133/695/295) do NOT reproduce numerically: standard DESeq2 on the deposited RSEM count tables gives 3-20x fewer DE genes at padj<0.05, and no single standard threshold matches; the data is biologically coherent (marker genes correct) but has high n=3 replicate dispersion and the paper's exact DE thresholds are not in the accessible Methods. NO fabrication indicated -- deposited data is internally consistent and biologically sensible; the gaps are reproducibility/under-specification, not data-integrity. NOT attempted (out of scope, wet-lab): LC-MS m6A, ELISA, proteomics MS, polysome westerns, qPCR decay half-lives, growth/sporulation assays.
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Assessment versions
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v1 current initial assessment Score 76assessed: 2026-06-19 ⛓ af11e021d832
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-29
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no 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 investigates the molecular function of the yeast m6A reader protein Pho92/Mrb1, testing how it recognizes m6A-modified meiotic mRNAs and links their translation to mRNA decay to promote meiotic fitness.
- ★ Pho92 specifically binds m6A-modified RNA via its YTH domain, both in vitro and in vivo finding
- ★ Pho92 associates with the 3' ends of meiotic mRNAs in both an m6A-dependent and m6A-independent manner (iCLIP/miCLIP) finding
- ★ Pho92 expression is developmentally regulated in early meiosis, driven by Ime1 and Ume6 binding at the PHO92 promoter finding
- ★ Pho92 transitions from the nucleus (via Paf1C interaction) to the cytoplasm, where it associates with translating ribosomes finding
- ★ Pho92 promotes and couples protein synthesis to mRNA decay of m6A-modified transcripts mechanism
- ★ Pho92-mediated mRNA decay is contingent on active translation and the CCR4-NOT complex mechanism
- Gis2 is enriched in m6A-oligo pulldowns but does not specifically bind m6A, unlike Pho92 finding
- ★ pho92 deletion decreases decay of m6A-modified mRNAs and impairs protein synthesis, reducing meiotic/gamete fitness finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RNA affinity pulldown + quantitative mass spectrometry (dimethyl labeling) | S. cerevisiae cell extracts (pre-meiosis and early meiosis, pCUP-IME1 synchronization) | m6A-modified vs unmodified RNA oligo bait | protein enrichment on m6A vs control bait | MS with light/heavy dimethyl isotope labeling |
| RT-qPCR and Western blot | diploid yeast, Pho92-V5 tagged strain | meiotic time course (SPO medium) | Pho92 mRNA and protein levels over time | — |
| ChIP-seq (reanalysis of published data) | S. cerevisiae, PHO92 locus | none | Ume6 occupancy at PHO92 promoter | — |
| Western blot | yeast, pCUP-IME1 Pho92-V5 strain | copper-sulphate induced IME1 expression | Pho92 protein accumulation | — |
| In vitro RNA binding / pulldown assay | recombinant GST-Pho92 and GST-Gis2 (bacterial expression), including N-terminal and YTH-domain truncations | m6A-modified vs control RNA oligo; YTHΔ and NΔ truncations | protein binding to oligos via SDS-PAGE/Coomassie/western blot | — |
| iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation), including 4TU-iCLIP for Pho92 | yeast, WT and ime4Δ, early meiosis (4 h SPO) | ime4Δ (loss of m6A methyltransferase) | Pho92 and Gis2 RNA binding sites/crosslink sites | — |
| miCLIP (m6A individual-nucleotide-resolution CLIP) | yeast, WT and ime4Δ, early meiosis (4 h SPO) | ime4Δ | m6A site mapping | — |
| RNA-seq (input libraries matched to CLIP) | yeast, WT and ime4Δ | ime4Δ | transcript abundance (TPM) to control for RNA level changes | — |
- ▲ Pho92 and Gis2 are enriched in binding to m6A-modified RNA bait over unmodified bait (log2 enrichment >2); Pho92 enrichment is specific to early meiosis while Gis2 occurs in both pre-meiosis and early meiosis log2 enrichment >2
- – Recombinant Pho92 binds m6A-modified oligo but not unmodified oligo; deletion of the YTH domain abolishes this binding while N-terminal deletion has no effect
- – Recombinant Gis2 does not bind m6A-modified or unmodified DRACH-motif oligos, but binds a control oligo with its own GA(A/U) motif
- ▲ Pho92 protein and mRNA are undetectable prior to Ime1 induction and rapidly accumulate after IME1 induction
- – PHO92 promoter contains a canonical Ume6 binding site ~230 nt upstream of the start codon with strong ChIP-seq signal 230 nt upstream
- – PCA of iCLIP/miCLIP counts per peak shows replicates cluster by genetic background (WT vs ime4Δ), with variance explained differing by protein/assay Gis2 34% vs miCLIP 80% vs Pho92 58% variance explained
- fold_change log2 enrichment >2 (threshold for calling Pho92/Gis2 enriched on m6A bait vs control in MS pulldown)
- fold_change log2FoldChange ≤ -1, adjusted p<0.001 (criteria for Ime4-dependent m6A sites in miCLIP)
- fold_change log2FoldChange ≤ -2, adjusted p<0.001 (criteria for Ime4-dependent binding sites in Pho92/Gis2 iCLIP)
- other 34% (Gis2), 80% (miCLIP), 58% (Pho92) (proportion of PCA variance explained by genetic background (WT vs ime4Δ) per assay)
- count 230 nt (distance of Ume6 binding site upstream of PHO92 start codon)
- count n=2 biological repeats (Pho92 RT-qPCR/western blot time courses (Figure 1D, 1F))
- count n=3 biological repeats (iCLIP and miCLIP experiments)
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.
The paper combines quantitative proteomics (differential dimethyl labelling with forward/reverse label-swap mass spectrometry) and iCLIP/miCLIP crosslinking approaches to map Pho92 and Gis2 RNA-binding sites, comparing wild-type to ime4Δ cells to distinguish m6A-dependent from -independent binding. Differential binding/methylation sites are defined using fold-change and adjusted p-value thresholds on volcano plots, reproducibility across replicates is assessed by PCA, and small-scale validation experiments (western blot, RT-qPCR) are reported from a small number of biological repeats without a named statistical test.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Differential enrichment/binding analysis reported as log2FoldChange with adjusted p-value (test/model not named in this excerpt) | Volcano plots comparing WT vs ime4Δ for miCLIP (Figure 1J) and Pho92 4TU-iCLIP (Figure 1K), used to call Ime4-dependent sites (log2FC ≤ -1, padj<0.001 for miCLIP; log2FC ≤ -2, padj<0.001 for iCLIP) | At least n=3 biological repeats stated for iCLIP/miCLIP experiments | not stated |
| Fold-change threshold on label-swap (forward/reverse) dimethyl-labelled MS intensities (log2 enrichment >2) | Scatter plot of m6A oligo pulldown vs control (Figure 1C, Figure 1—figure supplement 1B) | not stated | not stated |
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Ime4-dependent binding/methylation sites in miCLIP and iCLIP were defined using fixed log2 fold-change and adjusted p-value thresholds, with different fold-change cutoffs used for miCLIP versus iCLIP↳ Could also: A harmonized statistical framework (e.g., the same effect-size and FDR cutoff applied consistently across assay types, or a rank/percentile-based significance criterion) — Using consistent thresholds across related CLIP-based assays can make the relative stringency of site calling directly comparable between datasets
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Proteomic enrichment in the m6A pulldown was defined by a fixed log2 enrichment cutoff (>2) from forward/reverse label-swap mass spectrometry↳ Could also: A formal moderated statistical test (e.g., a moderated t-test such as limma) applied to the paired label-swap intensities, with multiple-testing correction — A formal test incorporating variance across replicate labelling reactions can complement a simple fold-change cutoff and provide a significance estimate alongside the enrichment magnitude
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Reproducibility among iCLIP/miCLIP biological replicates was assessed by visual clustering in principal component analysis↳ Could also: Reporting a quantitative reproducibility metric, such as pairwise Pearson or Spearman correlation coefficients between replicates — A numeric correlation statistic can complement PCA clustering by giving a directly comparable, quantitative measure of replicate concordance
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Western blot and RT-qPCR quantifications (e.g., Pho92 protein/RNA levels across a meiotic time course) are shown from n=2 biological repeats without an accompanying statistical test or dispersion measure↳ Could also: Increasing replicate number where feasible and reporting a paired comparison (e.g., paired t-test across matched time points) alongside SD, SEM, or individual data points — This would allow a formal statistical comparison between time points and convey the variability underlying the reported trend, which is useful when replicate numbers are small
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36422864 (Pho92 m6A reader, eLife 2022; van Werven/Ule labs)
Paper: Varier et al., eLife 2022, DOI 10.7554/eLife.84034. PMID 36422864 / PMC9731578. Code: https://github.com/ulelab/ncawareclip (Snakemake CLIP read-mapping pipeline). Data: GEO GSE193561 (SuperSeries) → 4 SubSeries, 73 GSM, S. cerevisiae, SRA PRJNA796594.
What the paper is
Multi-assay study of the m6A reader Pho92 in budding-yeast meiosis. Combines wet-lab (LC-MS m6A, ELISA, proteomics MS, polysome westerns, growth/sporulation assays) with several sequencing assays analysed by bioinformatic pipelines.
In-scope (pipeline-derived) results
Pipeline-derived results split into two layers:
Layer A — deposited pipeline outputs vs paper numbers (NO heavy compute needed)
GEO ships the processed binding-site BED files (the end products of the iCLIP/miCLIP analysis). Counting features in them directly tests whether the deposited data delivers the paper's headline counts:
- A1 Pho92 total binding sites (Fig 1K) —
GSE193291_Pho92_all.bed - A2 Pho92 Ime4-dependent sites (Fig 1K) —
GSE193291_Pho92_Ime4Dependent.bed - A3 m6A (miCLIP) sites (Fig 1J) —
GSE193291_m6A_allIme4Dependent.bed - A4 Gis2 total binding sites (Fig 1—S2F) —
GSE193291_Gis2_all.bed - A5 internal consistency: Ime4-independent = all − dependent (paper says 3171)
Layer B — regenerate from raw data on «our HPC» (heavy compute)
- B1 Peak→gene mapping: 507 genes (Pho92 Ime4-dep), 870 genes (m6A), 24 genes (Gis2 Ime4-dep). Needs bedtools intersect of deposited BEDs with R64/SGD gene annotation. (medium effort, no FASTQ)
- B2 Positional analysis: 23% (145/642) of Ime4-dep + 13% (410/3171) of Ime4-indep peaks at STOP codons (Fig 2E). Needs annotation + metagene.
- B3 Crosslink-track regeneration from FASTQ via ncawareclip (trim_galore → sequential bowtie/STAR ncRNA pre-mapping → genome STAR → crosslink bed). Repo ships ONLY human annotation prep ("currently human supported") → yeast annotation must be built first. Heaviest; full 1:1 from raw reads.
- B4 RNA-seq differential expression (GSE193558 ime1Δ/ndt80Δ; GSE193559 pho92Δ/ime4Δ): 295 up in pho92Δ, 1133 down in ime1Δ, 695 down in ndt80Δ, 212/505 Pho92 targets up in ime4Δ. Standard STAR/HISAT2 + DESeq2.
- B5 Overlaps (Fig 3A): Pho92∩Ime1=81, Pho92∩Ndt80=54 genes.
Out of scope (wet-lab / manual / external — NOT attempted)
- LC-MS m6A/A ratio (Fig 5A,G), m6A-ELISA — mass spec / biochemistry.
- Whole-proteome label-free MS, Pho92-IP MS (Fig 6E–I) — proteomics.
- Polysome-profiling westerns, growth/sporulation/viability assays — wet-lab.
- mRNA decay half-lives by qPCR time-course (Fig 5D) — wet-lab qPCR, not a sequencing pipeline (the t½ fit is trivial but the input is bench data).
- Re-used external datasets (Dierks 2021 m6A-seq2; Brar 2012 ribo-seq) — external.
- Motif (STREME) / YTFaSCo promoter motifs — attemptable if time (Layer B, lower priority).
Pipeline / tool inventory
- Demultiplex: Ultraplex (UMI in header
rbc:NNNN). Trim: trim_galore -q20 --length 11. - Map: ncawareclip — bowtie pre-map (rRNA/tRNA/snRNA/repeats) + STAR genome
(
--outFilterMultimapNmax 100), unique = MAPQ 255; crosslink = read start −1. - Peaks/significance: counts-per-peak → DESeq2; Ime4-dependent = log2FC ≤ −2 & padj < 0.001 (Pho92); m6A miCLIP threshold log2FC ≤ −1 & padj < 0.001.
- RNA-seq: aligner + DESeq2 (exact tool TBD from Methods; standard).
- Genome: R64-1-1 / sacCer3, 16 nuclear chromosomes (chr01–chr16 in deposited BEDs).
Approach decision
Layer A done locally (tiny deposited result files). Layer B on «our HPC»/«infra» when the central VPN is restored. P16 applies: running the authors' own pipeline + the deposited outputs is a fully valid reproduction.
Assessments & scoring basis
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.