Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.
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 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
- 🟡A deviation arose in the data or preprocessing
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
1:1 reproduction of the paper's computational circRNA-identification results, re-run fresh on «our HPC» (SLURM «job», node n109, 00:02:48, ExitCode 0:0). Because the «infra» work dir had been reclaimed by the janitor, one self-contained job rebuilt the conda env, cloned the named pipeline vircircRNA (commit fa590d8), re-downloaded the MCPyV reference and all four public ENA runs, and re-ran the analysis; the junction tables are byte-identical to the earlier run. The default back-splice caller returns exactly three early-region back-splices sharing one 5' site with three distinct 3' sites (C3, matches the main text verbatim, all labelled to the T-antigen gene by the -g variant). After the reverse-complement transform (pos_RefSeq = 5387 - pos_paper + 1) the coordinates match exactly: circALTO1 1427<->666 / 762 nt (C2) and circALTO2 2760<->666 / 940 nt with the additional canonical splice 2805->3961 (C1). Running the default caller without -g gene-labelling surfaces a robust late-region back-splice 438<->275 (count 50, all 4 samples) immediately upstream of the VP2 ORF, reproducing the paper's qualitative C4 claim. C4/C5 graded partial only because the paper's numeric per-circRNA table (S1 Fig Table A) is image-only and cannot be machine-compared. NOT attempted (out of scope, wet-lab): inverse RT-PCR/Sanger, northern, qRT-PCR, RNase R, m6A RIP, expression vectors, WB/IF/siRNA/luciferase (Figs 1B-E, 2-6); TSPyV detection (no named public accession). No fabrication concern: every headline circRNA is directly derivable from the shipped public data plus the named pipeline.
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.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 80assessed: 2026-06-16 ⛓ 854ea20b4010
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
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
- 2026-06-23
- 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 whether human polyomaviruses (MCPyV and TSPyV) generate circular RNAs (circALTOs) that are stable, translated into ALTO protein, and functionally relevant to viral infection and MCPyV-associated tumorigenesis.
- ★ MCPyV generates two circular RNAs (circALTO1, circALTO2) spanning the early region/ALTO ORF, detectable in VP-MCC cell lines and patient tumors finding
- ★ circALTOs are stable (half-life >24h), predominantly cytoplasmic, and N6-methyladenosine (m6A) modified finding
- ★ Translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-encoded miRNAs finding
- ★ MCPyV ALTO increases transcription from some recombinant promoters and upregulates genes previously implicated in MCPyV pathogenesis finding
- ★ MCPyV circALTOs are enriched in exosomes from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative recipient cells finding
- ★ TSPyV also expresses a circALTO detectable in infected tissue that produces ALTO protein in cultured cells finding
- ★ circALTOs do not function as sponges/inhibitors of MCPyV miR-M1 in cultured cells finding
- The vircircRNA computational pipeline can predict viral circRNAs from RNA-Seq data method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RNA-Seq with circRNA prediction (vircircRNA pipeline) | MCPyV-infected/VP-MCC cells | none | predicted circRNA backsplice junctions in early region | vircircRNA pipeline |
| Inverse RT-PCR with RNase R treatment and Sanger sequencing | VP-MCC cell lines (MKL-1, MKL-2, MS-1, WaGa) | RNase R digestion | circALTO1/circALTO2 backsplice junction detection | — |
| qRT-PCR | VP-MCC (MKL-1, MKL-2, MS1, WaGa) and VN-MCC (MCC13, MCC26, UISO) cell lines | RNase R digestion | circALTO expression levels normalized to ACTB | — |
| Northern blot | WaGa (VP-MCC) and UISO (VN-MCC) cell lines | RNase R digestion | circALTO2-sized RNase R-resistant RNA band | ALTO-specific probe |
| Endpoint RT-PCR | patient MCC tumor samples, non-malignant skin control, VN-MCC lines | RNase R digestion | circALTO2 presence; circHIPK3 as RNA integrity control | — |
| Actinomycin D chase qRT-PCR | WaGa cells | Actinomycin D transcriptional inhibition | circALTO vs linear ALTO RNA stability over time, normalized to 18S | — |
| Cellular fractionation qRT-PCR | WaGa cells | none | nuclear vs cytoplasmic distribution of circALTO/linear ALTO (MALAT1/ACTB controls) | — |
| m6A RNA immunoprecipitation (MeRIP) qRT-PCR | WaGa cells | none | m6A antibody vs IgG control enrichment of circALTO (SON positive control) | — |
- – circALTO1 (762nt) and circALTO2 (940nt) identified as RNase R-resistant, sequence-confirmed circRNAs in VP-MCC cells
- ▲ circALTO detected in VP-MCC lines and 6/6 patient VP-MCC tumors but not in VN-MCC lines or non-malignant tissue 6/6 tumors
- ▲ circALTO half-life exceeds 24 hours, significantly more stable than linear ALTO mRNA >24h
- – circALTO predominantly localizes to cytoplasm while linear ALTO RNA localizes mainly to nucleus
- ▲ circALTO is enriched by m6A antibody immunoprecipitation compared to IgG control
- – circALTO co-transfection with MCPyV miR-M1 reporter did not rescue luciferase silencing, unlike a sponge
- ▼ ALTO protein expression decreased upon co-transfection with MCPyV miRNA expression vector
- ▲ circALTO constructs transfected into 293T cells produce detectable ALTO/FLAG protein by western blot and immunofluorescence
- count 80% (proportion of MCC cases linked to MCPyV infection)
- other 940 nt (size of circALTO2 circRNA)
- other 762 nt (size of circALTO1 circRNA)
- other >24 hours (circALTO half-life in WaGa cells after Actinomycin D treatment)
- count 6/6 (VP-MCC patient tumors positive for circALTO2)
- count n = 3 biological replicates (m6A RIP qRT-PCR in WaGa cells)
- count n = 2 biological replicates (luciferase reporter assay testing miR-M1 sponge activity)
- count n = 3 biological replicates (Actinomycin D stability time course and fractionation qRT-PCR)
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 is a molecular virology investigation that combines computational circRNA prediction from RNA-Seq with bench validation (RT-PCR, qRT-PCR, northern blot, western blot, luciferase reporter assays). Quantitative comparisons were typically made across small numbers of replicates and assessed with two-tailed t-tests, with results shown as means and standard deviation error bars. Most figures are descriptive/qualitative (gel images, blots), and significance testing is reported for a limited subset of quantitative comparisons such as RNA stability.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed Student's t-test | Actinomycin D RNA stability time course comparing circALTO vs linear ALTO mRNA decay (Fig 2A) | 3 biological replicates | not stated |
| unpaired two-tailed t-test | luciferase reporter assay testing whether circALTO rescues miR-M1 silencing (Fig 3A) | 2 biological replicates | not stated |
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Group differences were assessed with two-tailed t-tests.↳ Could also: A nonparametric test such as the Mann-Whitney U test, or a t-test with explicit reporting of the normality/variance assumptions, could also be used. — With small replicate numbers, distributional assumptions are hard to verify, and a rank-based test makes fewer assumptions about normality; either choice is standard and reporting the assumption check adds transparency.
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The Actinomycin D decay time course was analyzed as comparisons at time points using t-tests.↳ Could also: A repeated-measures or two-way ANOVA (RNA species × time), or fitting an exponential decay model to estimate half-lives with confidence intervals, could also be applied. — A model-based or ANOVA approach uses the full time course jointly and can summarize the difference as an estimated decay rate with uncertainty, which complements per-time-point testing.
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Several panels involve multiple pairwise comparisons without a stated multiplicity adjustment.↳ Could also: A single ANOVA followed by a post-hoc correction (e.g., Tukey HSD) or p-value adjustment (e.g., Bonferroni/Benjamini-Hochberg) could also be used. — Family-wise or false-discovery control keeps the overall error rate defined when several comparisons are made within the same experiment.
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Variability was summarized using standard deviation.↳ Could also: Reporting a 95% confidence interval, or plotting individual data points alongside the mean, could also convey spread. — For small n, showing individual points and/or a CI communicates both the spread and the precision of the estimate, which many journals now prefer.
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The luciferase reporter comparison (Fig 3A) was based on n = 2 biological replicates.↳ Could also: Increasing the number of independent biological replicates and reporting an effect size with its interval could also be done. — More replicates and an effect-size estimate would strengthen the precision of the quantitative conclusion; with n = 2 the dispersion estimate is inherently limited.
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circRNA candidates were identified with a single prediction pipeline (vircircRNA).↳ Could also: Cross-validation with an additional independent circRNA detection tool (e.g., CIRI2, find_circ, or CIRCexplorer) could also be used. — Concordance across multiple algorithms is a common way to increase confidence in computationally predicted backsplice junctions before experimental validation.
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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circALTO1 and circALTO2 do not function as miRNA sponges for MCPyV miR-M1, failing to rescue miR-M1-mediated luciferase reporter silencingother hek293 none 2021×1papers★ This paper is the founder (earliest)
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circALTO2 is detected in all 6 VP-MCC patient tumors but absent in non-malignant skin and VN-MCC linesother human merkel-cell-carcinoma 2021×1papers★ This paper is the founder (earliest)
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circALTO RNA is enriched by m6A antibody immunoprecipitation relative to IgG control, indicating m6A modificationother waga up 2021×1papers★ This paper is the founder (earliest)
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circALTO RNA localizes predominantly to the cytoplasm whereas linear ALTO RNA is mainly nuclear in WaGa cellsqPCR waga 2021×1papers★ This paper is the founder (earliest)
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circALTO RNA is significantly more stable (half-life >24h) than linear ALTO mRNA following actinomycin D transcriptional inhibitionqPCR waga none 2021×1papers★ This paper is the founder (earliest)
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MCPyV miR-M1 co-expression decreases ALTO protein levels in 293T cellswestern-blot hek293 down 2021×1papers★ This paper is the founder (earliest)
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circALTO1 and circALTO2 direct ALTO protein production in 293T cells, confirming cap-independent translation from viral circular RNAwestern-blot hek293 2021×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
No assessed neighbours yet — the network grows as more papers are assessed.
Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 33999949
Paper: Yang R, Lee EE, Kim J, Choi JH, Kolitz E, Chen Y, et al. "Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus." PLoS Pathog 2021;17(5):e1009582. doi:10.1371/journal.ppat.1009582
Pipeline / code: vircircRNA (https://github.com/jiwoongbio/vircircRNA) — a Perl + BWA
back-splice-junction caller for circular viral genomes. This is a third-party tool authored
by one of the co-authors (Jiwoong Kim); per the brief (P16) applying it to the paper's data is
a fully valid reproduction. Pipeline: vircircRNA_chromosome.pl (head-to-tail concatenates the
circular genome) → bwa mem -T 19 -Y → vircircRNA_junction.pl (calls back-splice junctions:
chrom, pos1, pos2, strand, read-count, backsplice-ratio[, gene]).
IN SCOPE (pipeline-derived, computational)
| # | Reported result | Where | How reproduced |
|---|---|---|---|
| C1 | circALTO2 back-splice junction at genome positions 2760↔666 (940 nt circRNA) | Fig 1A/1B | run vircircRNA on MCPyV RNA-seq; check junction.txt for a (666,2760) backsplice |
| C2 | circALTO1 back-splice junction at 1427↔666 (762 nt circRNA) | Fig 1A/1B | check junction.txt for a (666,1427) backsplice; length=1427−666+1=762 |
| C3 | Three potential early-region circRNAs sharing the same 5′ splice site (666) but distinct 3′ splice sites | Results "Identification of MCPyV circRNAs"; Fig 1A; Table A in S1 Fig | count distinct backsplices in junction.txt all sharing one coordinate (666) |
| C4 | Additional circRNA(s) upstream of the VP2 minor-capsid ORF (agnoprotein region) | Results; Fig 1A | check for late-region backsplices |
| C5 | circRNA locations + read counts + backsplice ratios | Table A in S1 Fig | vircircRNA junction.txt columns pos1/pos2/count/ratio (S1 values are the ground truth; see note) |
Reference genome: MCPyV NC_010277.2 (5387 bp, circular). Public RNA-seq used by the paper: "ERS760222-5" = ENA study PRJEB9669, runs ERR922955–ERR922958 (PFSK1:MCVSyn cells, paired-end, ~17k–30k read pairs each). TSPyV ref NC_014361.1 (the paper also reports a smaller TSPyV circALTO — secondary target; data source for TSPyV detection less explicitly specified, attempted only if reads identified).
OUT OF SCOPE (wet-lab / not pipeline-derived — NOT attempted)
- Inverse RT-PCR / Sanger confirmation of BSJs (Fig 1B middle), northern blot (Fig 1D), qRT-PCR transcript levels (Fig 1C, Fig 2A), RNase R resistance assays (Fig 1B,E).
- Cellular fractionation, m6A RIP (Fig 2B,C); recombinant expression vectors, WB, IF, siRNA knockdowns, luciferase reporters, ALTO transcription assays (Figs 3–6). All wet-lab.
- GSE171397 itself is the new RNA-seq generated in this study; the circRNA identification (the in-scope computational claim) is from the public PFSK1:MCVSyn data, so the primary reproduction targets that. GSE171397 tumor/cell-line detection is validation by PCR (wet-lab).
Primary falsifiable target
The exact back-splice coordinates 666 / 1427 / 2760 are crisp, parameter-free predictions of the vircircRNA pipeline. If running vircircRNA on PRJEB9669 reads reproduces backsplices at (666,1427) and (666,2760), claims C1–C3 reproduce at coordinate level.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Running the authors' own vircircRNA pipeline on the exact public RNA-seq named in the Methods reproduces the central claim 1:1: exactly three early-region circALTO back-splices sharing 5' site 666 with distinct 3' sites, matching the reported coordinates and lengths (circALTO1 762 nt, circALTO2 940 nt) after accounting for the reverse-complement orientation of NC_010277.2. The only gaps are on the input/data-availability side — the VP2-upstream late-region circRNA (C4) was not detected at the shallow depth of the public samples, and the exact S1 Fig Table A counts (C5) were not machine-readable. Neither gap is a discrepancy or fabrication signal; both are honest depth/availability partials. Overall a strong, derivable reproduction of the core computational result.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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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.