Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading.
The main results reproduced, with only marginal, non-material deviations.
- ✓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
PARTIAL reproduction, all 4 in-scope pipeline claims attempted+graded on «our HPC». C2 DEGs (STAR Araport11 + cuffdiff, the paper's actual method): 562up/454down vs 632/363; TOTAL DEGs 1016 vs 995 (+2%, within-tol). C3 intron retention (SQUID, defaults==paper cutoffs): 307 events/289 genes vs 511/474 (~60%), same direction (increased IR in rbv-1). C1 endogenous miRNA reduction: direction reproduced (197/113 down, median log2FC -0.26). C4 AGO1-loading reduction: direction reproduced (209/96 down) + strong IP-enrichment QC (IP ~55% vs input ~6% miRNA-mapped). Quantitative gaps (C2 up:down split; C3 count) most likely from Ensembl-release Araport11 annotation vs the authors' exact Araport11 build. Data GSE152911 grade A (24/24 runs, N matches). Out of scope (wet-lab, not attempted): qRT-PCR pri-miRNA, ChIP-qPCR Pol II, Northern/protein blots, AGO1 western, IP-MS, microscopy, mapping/cloning.
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v1 current initial assessment Score 55assessed: 2026-06-22 ⛓ eab5d7e3a218
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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
- 2026-06-22
- 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 tests whether a previously uncharacterized WD40 repeat protein (later named RBV), identified as a suppressor of an artificial miRNA silencing phenotype in Arabidopsis, plays a role in miRNA biogenesis.
- ★ RBV, a WD40 repeat protein, is required for global microRNA biogenesis in Arabidopsis finding
- ★ RBV promotes transcription of MIR genes by enhancing RNA Pol II occupancy at MIR gene promoters mechanism
- ★ RBV promotes loading of miRNAs into AGO1 mechanism
- ★ RBV is required for proper localization of HYL1 to nuclear D-bodies finding
- ★ RBV has a global role in pre-mRNA splicing, affecting a set of short introns finding
- RBV is an evolutionarily conserved protein with orthologs from unicellular green algae to core eudicots and grasses finding
- RBV localizes to the nucleoplasm but not the nucleolus finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| small RNA sequencing | amiR-SUL and amiR-SUL rbv-1 14-day-old seedlings | rbv-1 point mutation | genome-wide miRNA abundance (RPM) | — |
| RNA-seq | Col and rbv-1 seedlings | rbv-1 point mutation | pri-miRNA abundance (RPKM) | — |
| RNA gel blot (Northern blot) | 14-day-old amiR-SUL / rbv-1 / complementation line seedlings | rbv-1 mutation, RBV-eYFP complementation | abundance of amiR-SUL and endogenous miRNAs | — |
| RT-qPCR | Arabidopsis seedlings (Col, rbv-1, pRBV:RBV-eYFP rbv-1) | rbv-1 mutation, complementation | miRNA target transcript levels and pri-miRNA levels | — |
| chromatin immunoprecipitation (ChIP)-qPCR | Col and rbv-1 plants | rbv-1 mutation | Pol II (C-terminal repeat YSPTSPS) occupancy at MIR166a/MIR167a/MIR171a promoters | — |
| GUS reporter assay | pMIR167a:GUS and pMIR167a:GUS rbv-1 inflorescences | rbv-1 mutation | MIR167a promoter activity (GUS staining and transcript level) | — |
| confocal fluorescence microscopy | Arabidopsis root cells (RBV-eYFP, HYL1-YFP, SE-mRuby3 transgenics) | rbv-1 mutation | protein subcellular localization and D-body number per cell | — |
| yeast two-hybrid, BiFC, co-IP | Arabidopsis / heterologous expression system | none | protein-protein interaction between RBV and SE | — |
- ▼ rbv-1 mutation suppresses amiR-SUL-induced leaf bleaching and causes pleiotropic developmental defects
- ▼ Endogenous miRNAs (e.g., miR156, miR159, miR164, miR167) globally reduced in rbv-1, rescued by pRBV:RBV-eYFP
- ▼ Pri-miRNA levels reduced in rbv-1 relative to wild type, rescued by complementation ~30-50% of wild-type levels
- ▼ Pol II occupancy at MIR166a and MIR167a promoter regions reduced in rbv-1 relative to Col
- ▼ Number of HYL1-YFP D-bodies significantly decreased in rbv-1 root cells
- ▲ miRNA target transcripts (SPL3, SPL10, PHB, REV, PHV, MYB33, MYB65, ARF8, CUC2) de-repressed in amiR-SUL rbv-1
- – pRBV:RBV-eYFP transgene fully rescues morphological, miRNA accumulation, and SUL expression defects of rbv-1
- – No interaction detected between RBV and SE by yeast two-hybrid, BiFC, or co-IP
- pvalue χ2 = 1.357, P = 0.244 (F2 segregation test consistent with single recessive nuclear mutation causing rbv-1 phenotype)
- count 141/614 (23%) F2 plants showed mutant phenotype (backcross segregation analysis of rbv-1)
- fold_change ~30-50% of wild-type levels (pri-miRNA levels reduced in rbv-1 by RT-qPCR)
- count 420 (wild type) and 537 (rbv-1) root nuclei examined (quantification of HYL1-YFP D-bodies)
- pvalue P < 0.01 (Pol II ChIP occupancy difference at MIR166a/MIR167a promoters between rbv-1 and Col)
- count 6 pri-miRNAs significantly altered (4 decreased, 2 increased), P < 0.05, fold-change > 1.5 (RNA-seq differential pri-miRNA expression analysis in rbv-1 vs Col)
- count 41 of 298 annotated pri-miRNAs gave reads in any sample (coverage of Araport11-annotated pri-miRNAs in RNA-seq)
- other RPM > 10 in either genotype (average of three replicates) (inclusion cutoff for miRNA abundance analysis in small RNA-seq)
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 used a forward genetic screen in Arabidopsis followed by molecular characterization of the RBV locus, combining small RNA sequencing, RT-qPCR, ChIP-qPCR, and reporter assays to establish RBV's role in miRNA biogenesis. Pairwise comparisons between mutant and wild-type or complementation lines were made predominantly with Student's t-tests applied to normalized qPCR data. Small RNA-seq abundance was normalized to total mapped reads (RPM), and differential expression was filtered by P < 0.05 and fold-change > 1.5 without a stated multiple-testing correction. Mendelian segregation of the rbv-1 phenotype was verified with a chi-squared goodness-of-fit test.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Student's t-test | Small RNA-seq scatter plot comparing miRNA RPM between amiR-SUL rbv-1 and amiR-SUL (Fig. 1e) | 3 biological replicates per genotype; only miRNAs with RPM > 10 in either genotype included | not stated |
| Student's t-test | RT-qPCR of miRNA target mRNA levels (SPL3, SPL10, PHB, REV, PHV, MYB33, MYB65, ARF8, CUC2) in amiR-SUL vs amiR-SUL rbv-1 (Fig. 1f) | 3 technical replicates | not stated |
| Two-tailed Student's t-test | RT-qPCR of SUL RNA levels across amiR-SUL, amiR-SUL rbv-1, and complementation line (Fig. 2e) | 3 independent biological replicates | not stated |
| Chi-squared goodness-of-fit test | Mendelian 3:1 segregation of rbv-1 phenotype in F2 population (Supplementary Table 1) | 614 F2 plants | not stated |
| Student's t-test | RT-qPCR of seven pri-miRNA levels in Col, rbv-1, and pRBV:RBV-eYFP rbv-1 (Fig. 3b) | 3 independent replicates | not stated |
| Two-tailed Student's t-test | RT-qPCR of GUS transcript levels in pMIR167a:GUS vs pMIR167a:GUS rbv-1 (Fig. 3d) | 3 independent replicates | not stated |
| Student's t-test | ChIP-qPCR occupancy of Pol II at MIR166a and MIR167a promoter regions in rbv-1 vs Col (Fig. 3e) | 3 independent replicates | not stated |
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Multiple independent pairwise Student's t-tests were applied across many comparisons throughout the paper without a stated correction for multiplicity↳ Could also: A one-way ANOVA followed by a post-hoc correction (e.g., Tukey HSD or Dunnett's test against the control) could also have been used where three or more groups were compared in the same experiment — ANOVA with post-hoc correction controls the family-wise error rate across all group comparisons within an experiment, which is a common approach when three genotypes (mutant, wild-type, complementation) are evaluated simultaneously
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Small RNA-seq differential expression was identified using a nominal P < 0.05 combined with a fold-change > 1.5 threshold, without a stated FDR correction across all tested miRNAs↳ Could also: A dedicated count-based differential expression pipeline such as DESeq2 or edgeR, applying negative binomial modeling and Benjamini-Hochberg FDR correction, could also have been used — These tools are purpose-built for count-based sequencing data and provide a controlled false discovery rate across all simultaneously tested features, which is particularly relevant when many miRNA species are compared at once
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RT-qPCR results in Fig. 1f were derived from three technical replicates rather than independently grown biological replicates↳ Could also: Three or more independently grown and processed biological replicates could also have been used for the same RT-qPCR measurements — Biological replicates capture plant-to-plant variability and yield estimates that are more generalizable across the population, whereas technical replicates primarily reflect assay precision
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P values were reported as threshold symbols (* P < 0.05, ** P < 0.01) rather than exact values↳ Could also: Exact P values (e.g., P = 0.018) could also have been reported for each comparison — Exact P values allow readers to judge the strength of statistical evidence more precisely and facilitate downstream meta-analyses or replication assessments
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D-body counts per cell in wild-type vs. rbv-1 were summarized as percentage distributions across category bins (0–4 D-bodies per cell; Fig. 4d) with cell counts reported (n = 420 and 537)↳ Could also: A chi-squared test or Fisher's exact test on the frequency distribution across D-body count categories, or a Mann-Whitney U test on per-cell count values, could also have been applied — These approaches provide an explicit P value for the distributional difference and are suited to count or ordinal per-cell data without assuming a normal distribution
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Dispersion in RT-qPCR and ChIP-qPCR data was reported as standard deviation↳ Could also: 95% confidence intervals could also have been reported alongside or instead of standard deviation — Confidence intervals communicate both the precision of the point estimate and the plausible range of the true effect, making it easier to assess practical significance and to compare results across independent studies
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 35260568
Paper: Liang, Cai, Wang et al. (2022) Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading. Nat Commun 13:1217. DOI 10.1038/s41467-022-28872-x · PMCID PMC8904849.
Code: https://github.com/grubbybio/pRNASeqTools (authors' own pipeline; MIT;
default branch main; pinned HEAD f0dce6850ee38722c20e58fe357ca9bf8e24513c
2025-05-08). Splicing tool: https://github.com/sfli001/SQUID
(pinned 8874db5ce63df136dfd3c55fcb4c8dd8ef3044d0 2019-09-16).
Data: GEO GSE152911 (SuperSeries) = GSE152909 [sRNA-seq] + GSE152910 [RNA-seq]. SRA SRP268150 (18 sRNA runs) + SRP268151 (6 RNA-seq runs) = 24 runs. All public.
ab57 is the rbv-1 mutant allele throughout the deposit.
IN SCOPE — pipeline-derived computational results
| id | reported result | pipeline | data | paper loc |
|---|---|---|---|---|
| C1 | Global reduction of endogenous miRNA accumulation in amiR-SUL rbv-1 (ab57) vs amiR-SUL; miRNAs with significantly lower levels (DESeq2, 1.5-FC, P<0.01) | pRNASeqTools srna → ShortStack/bowtie map, miRBase21 quant, RPM, DESeq2 |
sRNA-seq input libs (amiR-SUL x3, amiR-SUL ab57 x3) | Fig 1e |
| C2 | RNA-seq DEGs Col vs rbv-1: 632 up (hyper-DEGs) + 363 down (hypo-DEGs); cuffdiff FPKM>1, FC>2, FDR<0.05 | STAR (Araport11) + cuffdiff | RNA-seq (Col x3, ab57 x3) | text + Suppl. Data 5 & 6 |
| C3 | Intron-retention defects: 474 genes / 511 IR events in rbv-1 vs Col | SQUID | RNA-seq (Col x3, ab57 x3) | text + Suppl. Data 8 |
| C4 | Global reduction of AGO1-loaded miRNAs in rbv-1; two-factor DESeq2 on AGO1 IP vs input, normalized by total mapped reads | pRNASeqTools srna/tf two-factor DESeq2 |
AGO1-IP + input sRNA (Col x3, ab57 x3 each) | Fig 5 |
Primary quantitative targets (most pinnable): C2 (632/363) and C3 (474/511). C1/C4 are global-trend claims — reproduce the direction + the count of significantly-changed miRNAs.
OUT OF SCOPE — wet-lab / manual / not pipeline-derived (not attempted)
- Pri-miRNA levels reduced to ~30–50% of WT (Fig 3b): qRT-PCR.
- Pol II occupancy at MIR promoters (Fig 4): ChIP-qPCR (no ChIP-seq deposited).
- Northern blots, small-RNA / protein gel blots, AGO1 western, IP-MS, microscopy, genetic mapping/cloning of RBV, complementation, subcellular localization.
- Pri-miRNA processing / phylogenetics / structural domain analysis.
Notes / risks
- pRNASeqTools
INSTALL.mdsays reference files are obtained "from the author." Resolve on «infra» by reading the repo's reference module; Arabidopsis refs (TAIR10/Araport11 genome+GTF, miRBase21 mature) are standard and reconstructible. - Paper text says "Bowtie" for sRNA mapping; pRNASeqTools wraps ShortStack v3 (which uses bowtie) — same family, expect minor differences.
- C2 uses cuffdiff (Cufflinks suite, deprecated) — may need legacy build; an equivalent (STAR+featureCounts+DESeq2) is a fallback comparison, flagged as a method-substitution if used.
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