Decoding the MYC locus reveals a druggable ultraconserved RNA element

Name: Verified Rigor Record — Decoding the MYC locus reveals a druggable ultraconserved RNA element
Item: Decoding the MYC locus reveals a druggable ultraconserved RNA element
Rating: 86
Author: Αlpha¹

Shi, P.; Yang, F.; FNU, T.; Huang, W.; Aparicio, A. O.; Kalicki, C. H.; Trehan, A.; Murphy, M. R.; Rotlevi, E. R.; Xing, L.; Reilly, M. P.; Que, J.; Wu, X.

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Αlpha¹ Rigor Review

Rigor Review Verified

Critical

High-priority

Action items to resolve · Critical / High

Confidence: highEngine: 7.6.0Source: full text

Not verified in this report

Declared data/code links were not checked for liveness or content.

This Alpha1 Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper presents a technically rigorous CRISPR saturation mutagenesis screen of the MYC locus with robust experimental design and validation, but has several reporting gaps: missing data repository deposition for sequencing data, incomplete antibody and software version documentation, and no verification of statistical test assumptions.

Three independent reviewers evaluated all eight dimensions. Reviewers diverged on study design and statistical analysis (one 'warn' vs two 'pass' each), and on data code availability (one 'fail' vs two 'warn'). I resolved each by weighing the combined textual evidence. Ethical approvals and biological variables had conflicting not applicable/pass assessments which I resolved by the specific applicability of sub-criteria. The statistical verification component only checked 1 test; the majority of statistical analyses remain unverified.

12 major claims checked against the paper's own evidence: all adequately supported.

partialDiscussion, last paragraphReviewer 3
The findings provide 'a generalizable platform for base-pair-resolution functional dissection of endogenous genes'.
The paper demonstrates the platform works for MYC but does not test generalizability to other genes. This is a reasonable claim about the method's potential, not an overstatement.
Evidence: The paper shows the method works for one locus with detailed validation.
“establishes a generalizable platform for base-pair-resolution functional dissection of endogenous genes”
supportedResults, paragraph 8Reviewer 1
The majority of functionally essential base-pairs in the MYC locus are noncoding.
The paper provides direct evidence from the saturation screen showing noncoding regions account for 67% of significant base-pairs.
Evidence: Figure 2A and associated text: 'noncoding regions collectively harbor the majority (67%) of functional sites'.
“noncoding regions collectively harbor the majority (67%) of functional sites”
supportedResults, paragraph 11Reviewer 1
The phenotypic impact of noncoding sequences correlates inversely with evolutionary conservation.
The paper shows statistically significant inverse correlation using phastCons scores (P = 3.0×10−6).
Evidence: Figure 3A-B and text: 'noncoding base-pairs with significant phenotypes tend to be less conserved than noncoding base-pairs without significant phenotypes'.
“noncoding base-pairs with significant phenotypes tend to be less conserved than noncoding base-pairs without significant phenotypes (P = 3.0×10−6)”
supportedResults, paragraphs 13-15Reviewers 1, 2
An ultraconserved 3' UTR element is indispensable for MYC-dependent cancer cells.
The claim is supported by sgRNA validation, clonal deletions, plasmid complementation, and ASO experiments across multiple cell lines.
Evidence: Figures 4B-D, 4F-J, and validation assays show growth defects upon targeting the element.
“these convergent observations from bulk-mutated populations, clonal cell lines, and plasmid-expressed mutants validate the screen results and demonstrate that a highly conserved element within the MYC 3′ UTR is functionally required to promote cell proliferation.”
supportedResults, paragraphs 15-17Reviewer 1
Steric-blocking antisense oligos targeting this RNA element selectively eliminate MYC-addicted cancer cells by suppressing MYC function without reducing MYC abundance.
ASO treatments reduce viability in MYC-dependent lines but not in MYC-independent HEK293T, and RNA-seq shows MYC target genes downregulated without MYC expression change.
Evidence: Figures 4F-J, 5A-C, and 5D-F: viability assays, unchanged MYC mRNA/protein, and GSEA showing MYC target suppression.
“the conserved 3’ UTR element is a vulnerability of MYC-dependent cancer cells that can be selectively targeted by ASOs”
supportedResults, paragraphs 18-19Reviewer 1
The 3′ UTR element promotes perinuclear localization of MYC mRNA and efficient nuclear import of MYC protein.
Immunofluorescence and RNAscope show redistribution of MYC protein to cytoplasm and MYC mRNA away from nucleus upon ASO treatment or deletion.
Evidence: Figures 6A-D: IF and RNAscope images quantified show reduced nuclear MYC and increased cytoplasmic mRNA distance.
“the 3’ UTR element promotes nuclear localization of MYC protein.”
supportedResults, 'The majority of functional base-pairs are noncoding', Figure 2AReviewers 2, 3
The majority (67%) of functionally essential base-pairs in the MYC locus are noncoding.
The claim is directly supported by the data presented in Figure 2A, showing that 67% of significant base-pairs are noncoding.
Evidence: Figure 2A shows a pie chart or bar graph (described in text) indicating 33% coding, 67% noncoding among significant base-pairs.
“noncoding regions collectively harbor the majority (67%) of functional sites”
supportedResults, 'A paradox between conservation and function'Reviewer 2
Noncoding sequences in MYC have reduced vertebrate conservation but increased human-specific constraints.
This claim is supported by the CDF plots in Figure 3A-D showing statistically significant differences (P = 3.0e-6 for phastCons, and stronger JARVIS constraint for significant bps).
Evidence: Figure 3A-D with KS test p-values.
“noncoding base-pairs with significant phenotypes tend to be less conserved than noncoding base-pairs without significant phenotypes (Figure 3A, P = 3.0×10−6)”
supportedAbstract, Results, 'Controlling MYC activity without affecting abundance'Reviewers 2, 3
Steric-blocking ASOs targeting this RNA element selectively eliminate MYC-addicted cancer cells by suppressing MYC function without reducing MYC abundance.
Supported by Figures 5A-B (no change in MYC mRNA/protein), GSEA analysis showing MYC target repression (Figure 5D), and reduced viability in MYC-dependent cancer lines vs HEK293T (Figure 4F-J, 4K-L).
Evidence: Figure 5A-D, Figure 4F-L.
“steric-blocking antisense oligos targeting this RNA element selectively eliminate MYC-addicted cancer cells by suppressing MYC function without reducing MYC abundance”
supportedAbstract, Results, 'The 3' UTR element regulates MYC mRNA and protein localization'Reviewers 2, 3
The 3' UTR element promotes perinuclear localization of MYC mRNA and efficient nuclear import of MYC protein.
This is directly supported by immunofluorescence (Figure 6A-B) and RNAScope (Figure 6C-D) showing redistribution of MYC mRNA and protein upon ASO treatment or mutation.
Evidence: Figures 6A-D.
“this 3′ UTR element promotes perinuclear localization of MYC mRNA and efficient nuclear import of the short-lived MYC protein”
supportedResults, 'A paradox between conservation and function'Reviewer 3
Phenotypic impact of noncoding sequences correlates inversely with evolutionary conservation.
The paper shows a statistically significant inverse correlation: noncoding base-pairs with significant phenotypes are less conserved (P=3.0e-6).
Evidence: Figure 3A-C and associated text showing CDF of conservation scores.
“noncoding base-pairs with significant phenotypes tend to be less conserved than noncoding base-pairs without significant phenotypes (, P = 3.0×10⁻⁶)”
supportedResults, 'An ultraconserved 3’ UTR element indispensable for MYC-addicted cells'Reviewer 3
An ultraconserved RNA element in the 3' UTR is indispensable for MYC-dependent cancer cells.
The paper provides multiple lines of evidence: CRISPR screen phenotype, competitive growth assays, clonal cell line phenotypes, and plasmid-based rescue/knockout experiments.
Evidence: Figures 4B-D and associated text.
“we further derived two clonal cell lines carrying the 8-nt CACAACCT deletion in the MYC locus and observed significant growth defect compared to control”

Recomputed 1 test: 1 consistent, 0 inconsistent, 1 via agent-written checks.

1 integrity concern flagged (0 high).

Rigor

Eight Dimensions4 pass · 3 warn

12 major claims1 partial11 supported

4 copyedit issues flagged: mostly consistency, clarity.

Checked 89 references: 79 verified — 10 not checked.

Alpha¹ Rigor Action ItemsFinal pass before journal submission

This is a post-publication audit. The paper is scientifically sound but has significant reporting gaps that a reader should weigh: the absence of raw sequencing data in a public repository limits reproducibility, and the missing antibody catalog numbers and software versions complicate verification of key results. A correction or erratum should be considered to add a complete data availability statement with GEO/SRA accession numbers, a key resources table with antibody RRIDs and software versions, and a statement on statistical test assumptions.

1.
HIGHdata code
Deposit raw sgRNA sequencing data and RNA-seq data in a public repository (GEO or SRA) and include accession numbers in the manuscript.
Without deposited data, the study's core reproducibility is compromised; this is a standard expectation for genomics studies.
2.
HIGHdata code
Replace the vague data availability statement ('available on request') with a concrete statement specifying the repository and accession number(s).
A precise data access procedure is required for community reuse and peer review.
3.
HIGHreporting
Create a key resources table listing all antibodies with vendor, catalog number, clone, and RRID; list software tools with version numbers (MAGeCK, DESeq2, STAR, Bowtie, CRISPResso2).
Incomplete antibody and software documentation hinders reproducibility and verification of key experimental results.
4.
HIGHrigor
Add a statement on statistical test assumptions (normality, equal variance) for each validation experiment, or justify use of non-parametric methods.
Verification of assumptions is important for the validity of p-values from t-tests and ANOVAs used in validation experiments.
5.
HIGHrigor
Report effect sizes with confidence intervals for all key comparisons (e.g., mean differences between conditions in validation assays).
Effect sizes with CIs provide more information than p-values alone and are essential for meta-analysis.
6.
HIGHrigor
State whether all cell lines were STR-authenticated; if performed, cite the authentication service or provide a statement that they were not authenticated.
Cell line authentication is a common requirement for reproducibility; unspecificied status leaves a gap in resource validation.
7.
MEDIUMreporting
Define error bars in all figure legends (SD, SEM, or CI) and justify the choice.
Ambiguous error bar definitions can mislead readers about data variability.
8.
MEDIUMcopyedit
Standardize the Cas9 variant name to 'HF1-SpRY-Cas9' throughout the manuscript for consistency with the methods.
Inconsistent naming between 'high-fidelity SpRY-Cas9' in results and 'HF1-SpRY-Cas9' in methods can confuse readers.
9.
MEDIUMcopyedit
Reword the sentence 'Take the day that after 48 h infection as Day 0' to 'The day after the 48-hour infection period was designated as Day 0.'
Original phrasing is grammatically awkward and may cause misinterpretation.
10.
MEDIUMreporting
Clarify the rationale for the 3' UTR ultraconserved element: state that despite having the strongest positive phenotype score, it did not reach statistical significance (FDR=0.07) and discuss potential reasons.
The method-result mismatch flagged in the integrity check may confuse readers and should be explicitly reconciled.
11.
LOWreporting
Add a reporting guideline checklist (e.g., ARRIVE for preclinical studies) as a supplementary file.
Though not standard for this study type, a checklist can enhance completeness of methodological reporting.
12.
LOWcopyedit
Standardize all figure and supplementary figure references in the text with consistent parentheses style.
Inconsistent figure referencing detracts from professional presentation.

Full text (115,059 chars)Analyzed: Jul 7, 2026

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