A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport.
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
- ✓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 held under reproduction
- ✓Overall, the reproduction was clean
- Every checked point held up.
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
Described well enough -> faithful 1:1 reproduction of the deterministic, pipeline-derived statistics. Zenodo 4322750 deposits the authors' Big-FISH/Cellpose pipeline plus the cleaned end-of-pipeline per-cell feature table (notebook/features.csv, 54263 cells). Re-running the deposited notebook logic (pandas + scipy Welch t-test) on «our HPC» reproduces EXACTLY: N=54,263 cells; the 8 centrosomal interest genes; and the central Fig-3 result that puromycin (polysome dissociation) significantly reduces the centrosomal mRNA proportion for all 8 centrosomal mRNAs (p down to ~0) but not for controls, while cycloheximide has minimal effect. NOT attempted (out of scope / data not deposited): the upstream image-analysis steps (raw 4-channel smFISH images, Cellpose segmentation, Big-FISH spot detection) producing features.csv -- raw images are not in the deposit; and the 602/728-gene high-throughput screen and Drosophila ortholog experiments (separate data not deposited). This is a P16 reproduction of the deposited features through the deposited pipeline; grades provisional, human must confirm.
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v1 current initial assessment Score 96assessed: 2026-06-18 ⛓ 2557cc90a8db
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-18
- 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: opusDo human mRNAs encoding centrosomal proteins localize to centrosomes, and if so, what is the mechanism—specifically, is their localization dependent on translation and active transport of polysomes?
- ★ A total of eight human mRNAs (PCNT, NIN, BICD2, CCDC88C, CEP350, HMMR, ASPM, NUMA1) localize at centrosomes. finding
- ★ Centrosomal mRNAs localize at different cell-cycle stages with a finely regulated, choreographed translational program. finding
- ★ Localization of all eight centrosomal mRNAs requires translation and the nascent protein (puromycin-sensitive, cycloheximide-insensitive). mechanism
- ★ ASPM and NUMA1 mRNAs localize via active movement/transport of endogenous polysomes toward the centrosome at mitotic onset. mechanism
- ★ ASPM polysomes associate with microtubules and localize by motor-driven transport or microtubule pulling. mechanism
- ★ Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation, indicating a conserved family. finding
- ★ A high-throughput smFISH (HT-smFISH) method was developed to screen centrosomal protein-coding genes. method
- ASPM, NUMA1, and HMMR mRNAs localize to the centrosome at the same time as their encoded proteins. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| High-throughput smFISH (HT-smFISH) screen | HeLa cells stably expressing Centrin1–GFP | none | centrosomal mRNA enrichment / localization across interphase and mitosis | high-throughput spinning-disk confocal microscope (200 nm lateral, 600 nm axial; oligonucleotide pool of 92,000) |
| Low-throughput smiFISH (validation) | HeLa cells stably expressing Centrin1–GFP | none | confirmation of centrosomal mRNA localization (Cy3 probes) | wide-field microscopy |
| smFISH against GFP RNA | HeLa Kyoto BAC cell lines (ASPM-GFP, NUMA1-GFP, HMMR-GFP) | none | mRNA localization at mitotic phases correlated with protein localization | 44 Cy3-labeled oligonucleotide probes |
| Two-color smFISH | HeLa BAC-GFP cell lines | none | pairwise co-localization of ASPM, NUMA1, HMMR mRNAs (Cy3 BAC-GFP mRNA + Cy5 endogenous mRNA) | — |
| Time-lapse live-cell microscopy | HeLa Kyoto BAC cell lines (ASPM-GFP, NUMA1-GFP, HMMR-GFP) | none | protein expression and localization across the cell cycle | — |
| smFISH with translation-inhibitor drug treatment | HeLa cells expressing Centrin1–GFP | drug (puromycin or cycloheximide, 5–20 min) | fraction of cells with localized centrosomal mRNAs | automated spinning-disk microscope, 63× oil objective |
| Automated high-content smFISH quantification | HeLa–Centrin1–GFP cells in 96-well plates | puromycin / cycloheximide | fraction of cells with localized mRNA, mitotic-phase classification by DAPI | automated spinning disk microscope, 3D imaging, automated DAPI classifier |
| smFISH in Drosophila (ortholog localization) | Drosophila (orthologs Girdin/Bsg25D/BICD/Plp/Asp/Mud/Cen/Cp110) | translation inhibition | centrosomal mRNA localization and translation dependence | — |
- – Eight human mRNAs localize at centrosomes; six newly confirmed (NIN, BICD2, CCDC88C, CEP350, plus PCNT and NUMA1) added to previously known ASPM, HMMR. 8 mRNAs
- – CCDC88C localizes in interphase but not mitosis; PCNT, NIN, BICD2, HMMR, CEP350 localize in interphase and early mitosis then delocalize; NUMA1 and ASPM localize only during mitosis.
- ▼ Puromycin delocalized all eight centrosomal mRNAs while cycloheximide had no effect.
- ▼ A 5-min puromycin incubation was sufficient to inhibit centrosomal localization of ASPM, NUMA1, and HMMR mRNAs at all mitotic phases where they normally localize.
- – ASPM-GFP mRNA and protein enriched together on mitotic centrosomes across all phases of division; NUMA1 and HMMR mRNAs accumulated only in prophase/prometaphase.
- – HMMR centrosomal mRNA/protein re-established at telophase cytokinetic bridges, unlike NUMA1.
- – The three mRNAs (ASPM, NUMA1, HMMR) never perfectly co-localized on centrosomes at any cell-cycle stage, occupying distinct peri-centrosomal regions.
- – Drosophila orthologs of the human centrosomal mRNAs localized to centrosomes in a translation-dependent manner.
- count 602 genes (genes encoding centrosomal/mitotic spindle proteins screened by HT-smFISH)
- count 8 mRNAs (human mRNAs localizing at centrosomes)
- count >40,000 cells (automated analysis of cells for quantitative validation)
- count 92,000 oligonucleotides (complex oligonucleotide pool used to generate probes)
- count 50–100 probes (individual probes designed against each mRNA)
- pvalue <0.0001 (****) (two-sided Fisher's exact test for centrosomal localization significance)
- count 44 Cy3 probes (oligonucleotide probe set against GFP RNA sequence)
- other 200 nm lateral, 600 nm axial (3D imaging resolution in the screen)
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 study is a high-throughput smFISH screen of 602 centrosomal-protein-coding genes in HeLa cells, followed by lower-throughput validation experiments and drug-treatment assays. The primary inferential method is the two-sided Fisher's exact test applied to proportions of cells showing centrosomal mRNA localization across cell-cycle phases. Results are presented as bar graphs of per-phase localization frequencies with 95% Wilson/Brown binomial confidence intervals and asterisk-coded significance tiers; no explicit multiple-testing correction is described for the series of pairwise comparisons performed.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Two-sided Fisher's exact test | Pairwise comparisons of the proportion of cells showing centrosomal mRNA localization across cell-cycle phases (Figs. 1c, 2b, 2d, 2f and related supplementary figures) | Total cell counts per bar from three independent experiments (exact per-bar N indicated in bar graphs but not reported numerically in the provided text); automated analysis used >40,000 cells | not stated |
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Multiple two-sided Fisher's exact tests were applied across the family of cell-cycle-phase comparisons within each mRNA, and across multiple mRNAs, without a stated multiplicity correction↳ Could also: Apply a false-discovery-rate procedure (e.g., Benjamini–Hochberg) or a family-wise correction (e.g., Bonferroni) across the set of pairwise tests within each figure panel — With many simultaneous comparisons, a multiplicity correction would make the claimed significance thresholds directly interpretable at the family level; readers could then distinguish which contrasts are robust after accounting for the number of tests
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P-values are reported only as asterisk tiers (<0.0001, ~0.0001, <0.01, <0.05) rather than as exact values↳ Could also: Report exact p-values (e.g., p = 0.0032) alongside or instead of symbol tiers — Exact p-values allow readers and future meta-analysts to apply their own significance thresholds, perform power calculations, and combine results across studies more precisely
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The magnitude of the difference in localization frequency between cell-cycle phases is not quantified beyond the p-value↳ Could also: Report an odds ratio (or relative risk) with its 95% CI for each pairwise comparison — An effect-size estimate would convey the practical magnitude of the enrichment at centrosomes, which is complementary information to the test of whether the difference is non-zero
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Proportions of localizing cells from three independent biological experiments were pooled and compared with Fisher's exact test↳ Could also: Model experiment-level variation with a mixed-effects logistic regression or a generalized linear mixed model treating experiment as a random effect — When counts are pooled across independent biological replicates, between-experiment variability is not captured; a mixed-effects approach would propagate that variance into the inference, which can matter when replicate-level proportions differ
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For the drug-treatment comparisons (puromycin vs. cycloheximide vs. control), the analysis appears to involve separate Fisher's exact tests per condition↳ Could also: Use a single chi-square test of independence (or log-linear model) across the three treatment groups simultaneously, followed by post-hoc pairwise contrasts — A single omnibus test controls the type-I error for the three-way comparison before drilling into pairs; chi-square and Fisher's exact tests are asymptotically equivalent at large n and the omnibus approach is the conventional framing for multi-group categorical data
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Centrosomal localization was scored as a binary outcome (localized / not localized) per cell↳ Could also: Quantify a continuous enrichment metric (e.g., mRNA spot density within a defined radius of the centrosome marker relative to cytoplasm) and compare groups with a non-parametric rank test (e.g., Mann–Whitney U) or a permutation test — A continuous enrichment score captures gradations in localization strength that a binary call may miss, and can be more sensitive for detecting partial or transient localization differences
What was reproduced
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Assessments & scoring basis
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a faithful 1:1 reproduction of the deterministic, deposited end-of-pipeline statistics: N=54,263 cells matches exactly, the 8 centrosomal genes match 8/8, and the Fig-3 puromycin result (significant centrosomal-localization loss for all 8 interest genes, ns controls; CHX minimal) reproduces exactly from features.csv via the shipped scipy Welch-test logic. All values are derivable from the shared data, with no fabrication signal. The only limitation is scope, not correctness — raw images, the 602/728-gene screen, and Drosophila ortholog experiments were not deposited and were not attempted (a q1/q2 availability matter, not an authors' defect on the reproduced claims).
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