Daily temperature cycles promote alternative splicing of RNAs encoding SR45a, a splicing regulator in maize.
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
- 🟡A deviation arose in the data or preprocessing
- 🔴A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡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
Described well enough to reproduce. The brief's 'code' (BGI-flexlab/SOAPnuke) is a generic FASTQ cleaner, not analysis code -> P16 reproduction with the named pipeline (HISAT2 + rMATS) on the paper's own data. The AS results live in GSE167670, not the brief's GSE154373 (the bZIP60/UPR companion deposit; same raw reads, different focus). TIER 1 (deposited-table audit, DONE): the paper's headline AS numbers reproduce well against the deposited GSE167670 tables -- per-sample AS events ~9476(V4)/~12018(V5) vs ~9000/~11000, AS genes 4756/5604 vs ~4700/~5600, and V4 DAS total = 544 EXACT with the correct monotone 62<181<301 increase across 33/35/37C. ONE CONCRETE DEFECT: the V5 Differential-AS supplementary table has its 37/31C block byte-identical to its 35/31C block (both 314 rows), inflating the deposited V5 DAS total to 775 vs the reported 691 and contradicting the paper's statement that 37/31 has fewer DAS than 35/31; the gap 775-691=84 implies a true 37/31 count near 230. ES+IR are the two dominant AS types (confirmed) though their DAS fraction (61-78%) runs higher than the stated 54-61%. TIER 2 (independent HISAT2+rMATS rerun on 24 raw FASTQ) is IN PROGRESS on «our HPC». NOT attempted: wet-lab RT-PCR/protoplast splicing assays and SR45a domain experiments (non-computational).
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 76assessed: 2026-06-18 ⛓ 38ac104c10ac
✎ 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
- 2026-06-18
- 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: opusThe study tests whether elevated and daily-cycling temperatures, simulating hot summer field conditions, alter the frequency and pattern of alternative RNA splicing in maize, and specifically whether daily temperature cycles change the RNA isoforms of the splicing regulator SR45a to encode proteins with greater splicing efficiency.
- ★ Increasing maximum daily temperature (MDT) globally elevates the frequency of alternative splicing in maize, particularly intron retention and exon skipping. finding
- ★ Genes most frequently targeted by increased AS at elevated MDT encode factors involved in RNA processing, plant development, and protein modification/chromosome segregation. finding
- ★ Genes encoding splicing regulators (e.g. SR45a) are themselves among the principal AS targets and show the most highly upregulated AS with increasing MDT. finding
- ★ Daily temperature cycles change SR45a RNA isoform abundance across the day, producing nonproductive isoforms in the virtual morning and potentially productive (full-length protein-encoding) isoforms in the warm virtual afternoon. mechanism
- ★ An 'in protoplast' RNA splicing assay was established to test the splicing efficiency of proteins encoded by different SR45a RNA isoforms on model substrates and to define the exonic splicing enhancers used. method
- ★ SR45a RNA isoforms produced later in the day at higher temperatures encode proteins with greater RNA splicing efficiency on model substrates. finding
- ★ AS changes are largely independent of transcriptional regulation, since few DAS genes were differentially expressed and heat-induced AS fold changes greatly exceeded expression fold changes. finding
- AS frequency declined with increasing MDT for some genes, such as a group encoding phosphatidylethanolamine-binding (FT-homolog) proteins. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq with alternative splicing analysis | maize (Zea mays) first fully expanded leaf at developmental stages V4 (20 DAG) and V5 (27 DAG) | daily temperature cycles with maximum daily temperatures of 31°C, 33°C, 35°C, or 37°C in the Enviratron | frequency and type of AS events (IR, ES, MXE, A5SS, A3SS) and differential alternative splicing (DAS) | Enviratron controlled-environment system (Bao et al., 2019) |
| gene expression / differential expression analysis (from RNA-seq) | maize leaf at V4 (20 DAG) and V5 (27 DAG) | elevated MDT (31–37°C) | transcript abundance / log2 fold change of splicing factor and regulator genes | — |
| GO enrichment analysis | maize alternatively spliced genes | increasing MDT | enriched biological processes among DAS genes | — |
| 'in protoplast' RNA splicing assay | maize protoplasts | overexpression of different SR45a RNA isoforms | splicing efficiency of model RNA substrates and identification of exonic splicing enhancers | — |
- ▲ ES and IR were the two major AS types, together contributing 54%–61% of total DAS events in leaves at higher MDT versus 31°C MDT. 54%–61%
- ▲ 544 DAS events at V4 (20 DAG) and 691 DAS events at V5 (27 DAG) identified relative to 31°C MDT. 544 and 691 DAS events
- – ~9,000 AS events across ~4,700 genes at V4, and ~11,000 AS events across ~5,600 genes at V5. ~9,000/~4,700 and ~11,000/~5,600
- ▲ Heat-induced expression of SR45a and SR34 was about seven-fold, but heat-induced AS at certain sites exceeded 1,000-fold (log2FC > 10). >1,000-fold AS vs ~7-fold expression
- ▲ Only four splicing regulator genes (SR45a, an mRNP protein, SCL33, SR34) were moderately upregulated by elevated MDT, less than eight-fold (log2FC < 3). <8-fold (log2FC < 3)
- – 89 of 679 DAS genes experienced multiple types of AS. 89 of 679
- ▲ Higher MDTs led SR45a to produce RNA isoforms capable of encoding full-length proteins from an early translation start site, whereas lower-MDT isoforms had the ORF blocked by PTCs.
- – Greatest increase in AS of splicing regulators occurred comparing 35°C/31°C MDT at V5 rather than 37°C/31°C, possibly due to plant weakening at chronic 37°C.
- count ~9,000 AS events out of ~4,700 genes (V4 stage (20 DAG))
- count ~11,000 AS events out of ~5,600 genes (V5 stage (27 DAG))
- count 544 and 691 DAS events (V4 and V5 vs 31°C MDT)
- other 54%–61% (ES+IR fraction of total DAS events at elevated MDT)
- fold_change log2FC > 10 (>1,000-fold) (heat-induced AS at certain sites for SR45a and SR34)
- fold_change ~seven-fold (heat-induced expression of SR45a and SR34)
- fold_change log2FC < 3 (<eight-fold) (upregulation of four splicing regulator genes by elevated MDT)
- count 89 out of 679 (DAS genes with multiple types of AS)
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 RNA-seq study examined differential alternative splicing (DAS) in maize leaves across four maximum daily temperature (MDT) conditions (31°C, 33°C, 35°C, 37°C) at two developmental stages (V4, V5), using three biological replicates per condition. DAS events were identified by applying a combined threshold of |Δψ| > 5% and FDR ≤1%, while differential gene expression was assessed with Q value ≤ 0.05 and |log2 FC| > 1. Results were summarized as counts of DAS events, log2 fold changes, and proportional Venn diagrams, supplemented by GO enrichment analysis of DAS-affected genes.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Percent-spliced-in (Δψ) differential splicing test with FDR control | All pairwise DAS comparisons of higher MDTs vs. 31°C MDT at stages V4 and V5 | 3 biological replicates per condition | not stated |
| Differential gene expression test with FDR/Q-value control | Gene expression comparisons across MDT conditions (Q ≤ 0.05, |log2FC| > 1) | 3 biological replicates per condition | not stated |
| Gene Ontology (GO) enrichment analysis | Genes showing increased DAS at higher MDTs (∼900 events) | — | not stated |
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Each higher MDT was compared pairwise to the 31°C reference in separate tests, with FDR applied within each comparison independently↳ Could also: A single multi-factor linear model (e.g. incorporating temperature as a continuous or ordered factor) could also be fitted across all four MDT levels simultaneously — A joint model would estimate a dose-response relationship across the temperature gradient and control the family-wise error rate across all MDT comparisons in one step, potentially increasing power and interpretability
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Dispersion around means in bar graphs was reported as SD with n=3 biological replicates↳ Could also: 95% confidence intervals or SEM could also be displayed — With n=3, the CI or SEM directly conveys uncertainty about the estimated mean and can facilitate visual inference; SD describes sample spread but does not directly reflect estimation precision at small n
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A minimum |Δψ| > 5% threshold was applied alongside FDR ≤1% as the criterion for calling DAS events↳ Could also: Reporting the full distribution of Δψ values (e.g. histogram or volcano plot of Δψ vs. –log10 FDR) across all events would also be informative — A distributional view would show whether effects cluster near the 5% threshold or include many large-magnitude changes, aiding interpretation of biological relevance beyond binary classification
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GO enrichment was used to characterize DAS-affected genes by biological process↳ Could also: Pathway-level analyses (e.g. KEGG, MapMan, or plant-specific ontologies) or network enrichment approaches could also be applied — Complementary databases capture functional groupings not well represented in GO (e.g. metabolic pathways, stress-response modules), potentially revealing additional biological context
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DAS and differential gene expression were analyzed as separate, independent outcomes↳ Could also: A joint or integrated splicing-expression model (e.g. LeafCutter with covariates, or a model explicitly testing splicing changes after regressing out expression level) could also be used — Explicitly modeling both simultaneously would more directly address the paper's key question of whether AS changes are independent of transcript abundance changes, rather than relying on post-hoc overlap comparisons
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Sample size of n=3 biological replicates per condition was used without a stated power calculation↳ Could also: A prospective power analysis for RNA-seq differential splicing (e.g. using tools such as RnaSeqSampleSize or simulations based on pilot data) could also accompany the design — A power estimate would clarify the minimum detectable Δψ effect size at a given FDR, helping readers interpret negative findings and assess the completeness of the DAS catalog
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 33705553 (maize SR45a alternative splicing)
Title: Daily temperature cycles promote alternative splicing of RNAs encoding SR45a, a splicing regulator in maize. Plant Physiol 186(2):1318–1335 (2021). PMCID PMC8195531 · DOI 10.1093/plphys/kiab110.
The "code" link is a generic read-cleaner (P16 reproduction)
Brief Code: = github.com/BGI-flexlab/SOAPnuke = a FASTQ quality-filter the BGI
sequencing service ran on the raw reads — NOT the authors' analysis code. No
authors' analysis repository exists. Same SOAPnuke red-herring pattern seen in
several other rooms (see kartei repro-pmid-36855106-sepsis-ceRNA,
repro-pmid39367086-lactylation-sepsis). => reproduce the pipeline-derived AS
results with the standard tools the Methods name, on the paper's own public data.
Data
- GSE167670 (this paper's AS reanalysis) — 24 SE RNA-seq samples, BGIseq500,
50 bp. Design:
E_W22_*= stage V4 (20 DAG),L_W22_*= stage V5 (27 DAG); each at MDT 31/33/35/37 °C × 3 reps = 24. SRA SRP308392 / PRJNA705177 (~140 GB). Each GSM is a "Reanalysis of" a GSM in GSE154373 → same raw reads as GSE154373. - GSE154373 (companion: bZIP60/UPR/HSR paper) — the brief's
Data:accession. Title does NOT match this paper; it is the transcriptional-DE deposit. The AS numbers come from GSE167670, not GSE154373. - GSE167670 also ships the processed result tables as supplementary files: per-sample AS-events CSVs (20/27 DAG) and Differential-AS (DAS) xlsx (20/27 DAG).
Pipeline (Experimental procedures)
SOAPnuke v1.5.2 (filter) → HISAT2 v2.0.4 (map to B73 reference genome) → Bowtie2 v2.2.5 + RSEM v1.2.12 (expression) → DESeq2 v1.4.5 (DE, Q≤0.05) → rMATS v3.2.5 (AS: ES/IR/A5SS/A3SS/MXE). DAS threshold |Δψ| > 5% and FDR ≤ 1%. Comparisons are each higher MDT vs 31 °C: 33/31, 35/31, 37/31, separately for V4 and V5.
In scope (pipeline-derived, reproduced)
- Tier 1 — verify deposited tables vs paper (DONE): per-sample AS-event counts (~9 000 V4 / ~11 000 V5), AS-gene counts (~4 700 / ~5 600), AS-type distribution, DAS totals 544 (V4) / 691 (V5), ES+IR fraction, multi-type-gene count.
- Tier 2 — independent rerun (HISAT2 + rMATS-turbo on the 24 FASTQ): regenerate the DAS counts from raw reads. Tool-version drift expected (rMATS 3.2.5 → turbo, HISAT2 2.0.4 → current, B73 v4 assembly) so grade on direction/order of magnitude.
Out of scope (not pipeline-reproducible here)
- All wet-lab results: RT-qPCR / semi-quant RT-PCR isoform validation, maize protoplast transient splicing assays, SR45a domain assays (Figs after Fig 1's AS overview). Manual / experimental, not computational.
- RSEM/DESeq2 DE gene lists (transcriptional) — secondary to the AS story; the paper emphasises that few DAS genes are DE. Could be added but not the headline.
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.
Against the authors' own deposited GSE167670 tables the paper's headline AS numbers reproduce well: V4 DAS=544 exact, the monotone 33<35<37C increase is confirmed, and per-sample AS-event/AS-gene counts match within ~9% rounding. The one substantive problem is authors'-side, not ours: the deposited V5 DAS table has its 37/31C block byte-identical to the 35/31C block, so the deposit sums to 775 instead of the reported 691 and contradicts the paper's own 'fewer DAS at 37/31' narrative. Severity is moderate — the core temperature-promotes-AS conclusion still holds at V4 and the V5 anomaly is a corrupt-deposit artifact (the implied true count ~230 is consistent with the paper text), with a secondary ES+IR fraction running above the stated 54–61%. The SR45a regulator claims are wet-lab and not reproduced.
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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.