IRES-TrAPPr reveals novel insights into viral and cellular mRNA translation

Name: Verified Rigor Record — IRES-TrAPPr reveals novel insights into viral and cellular mRNA translation
Item: IRES-TrAPPr reveals novel insights into viral and cellular mRNA translation
Rating: 86
Author: Αlpha¹

May, G. E.; McManus, J.

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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

References were not verified against Crossref/OpenAlex.
No reported statistical tests were found to recompute.

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 novel, well-validated RNA-based MPRA (IRES-TrAPPr) with strong scientific premise and robust study design, including appropriate controls and replication. Key reporting gaps include missing cell line authentication, incomplete code sharing, and insufficient statistical detail (exact p-values, effect sizes, software identification), which warrant consideration by readers but do not invalidate the primary findings.

Three independent reviewers evaluated the paper; two classified it as computational, one as interventional (we adopted computational). Divergences occurred in study design (pass vs. warn), biological variables (N/A/warn/pass), key resources (warn/pass), data code availability (warn/pass), and reporting transparency (warn/pass). Our synthesis weighs the combined evidence, leading to pass/improvement statuses. Statistical verification was not possible (0 tests recomputable, see coverage note).

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

partialResults, paragraph on Type VI IRESes and DiscussionReviewers 1, 3
Viral IRESes from warm-blooded hosts have adapted higher structural stability to maintain folding at higher temperatures.
The paper shows that Type IV IRESes from endothermic hosts are generally more active in human cells than those from ectothermic hosts, and that active IRESes have higher GC content. The sample size is limited, and the statistical significance is borderline for some comparisons (e.g., 'approached significance' for Type VIab).
Evidence: Comparison of GC content and activity: 'Active IRES elements had more GC pairs than inactive ones, though this only approached significance for Type VIab IRES elements and was not statistically significant for Type VIb'. Also, IRESes from ectothermic hosts were mostly inactive in human cells.
“These results suggest that Type IV IRESes from viruses infecting warm blooded animals may have evolved to be more structurally stable at higher temperatures.”
partialResults, 'Determining the relative activities of hundreds of viral IRESes' sectionReviewer 2
Viral IRESes from warm-blooded hosts have higher structural stability (more GC base pairs) and are more active in human cells.
The evidence shows a trend for Type IV IRESes (significant) but only a non-significant trend for Type VI IRESes; the claim is stronger for Type IV.
Evidence: Active IRES elements from Type IV had more GC pairs than inactive ones, and IRESes from endothermic hosts were generally more active than those from ectothermic hosts.
“Active IRES elements had more GC pairs than inactive ones, though this only approached significance for Type VIab IRES elements and was not statistically significant for Type VIb.”
supportedAbstract; Results, paragraph 1Reviewer 1
IRES-TrAPPr is a sensitive and specific method for measuring IRES activity and comparing it to cap-dependent initiation.
The paper provides comprehensive validation: high reproducibility (Pearson >0.99 technical, >0.94 biological), orthogonal luciferase validation of 21 candidates, and structure-function analysis recapitulating known IRES features.
Evidence: Demonstrated high reproducibility between replicates, validated by independent luciferase assays, and scanning mutagenesis experiments that replicate known functional elements.
“Using stringent positive and negative controls, we show this method is highly specific and corresponds to orthogonal expression assays with high precision.”
supportedResults, paragraph on evaluating viral and cellular IRES candidatesReviewer 1
The vast majority of short viral and cellular IRES elements proposed using DNA-based assays are false positives.
The paper shows that nearly all candidates from IRESbase (499 viral, 646 cellular) had negligible IRES activity in IRES-TrAPPr, with specific validation for several candidates. This directly supports the claim.
Evidence: Only 4 viral IRESes out of 499 tested were active; 0 cellular candidates showed clear activity. Additional validation via luciferase and Tornado circRNA systems.
“Surprisingly, only two viral IRESes from IRESbase were categorized as 'active' by IRES-TrAPPr... we found negligible activity from cellular IRES candidates, including all of the candidate 5’ UTR IRESes reported in the original bicistronic MPRA screen.”
supportedResults, paragraph on evaluating viral and cellular IRES candidatesReviewer 1
Zika and Dengue virus 5' UTRs have negligible IRES activity.
Multiple lines of evidence are provided: IRES-TrAPPr showed no activity, luciferase validation confirmed this, and experiments under stress conditions (HRV 2A protease, chemical inhibition) did not induce IRES activity. Tests in insect cells also showed no activity.
Evidence: IRES-TrAPPr, luciferase assays, HRV 2A stress experiments, chemical inhibition (rapamycin, 4EGI-1), and tests in mosquite (Aag2) and Drosophila (S2) cells.
“These results show that the Zika and Dengue 5’ UTRs have negligible IRES activity.”
supportedResults, paragraph on stress experimentsReviewers 1, 2, 3
IRES-TrAPPr can detect stress-dependent changes in CDI and IRES activity.
The paper demonstrates that thapsigargin treatment decreases CDI TE (~40%) and increases IRES activity for positive controls, while inactive candidates showed no change. This is supported by polysome profiling and spike-in controls.
Evidence: Polysome profiles showing increased monosome signal under thapsigargin; TE estimates showing decreased G-cap and increased A-cap for active IRESes.
“Our TE estimates from IRES-TrAPPr revealed that CDI reporters had an ~40% decrease in translation efficiency in response to thapsigargin treatment... our positive control IRES sequences showed increased translation efficiency in response to stress.”
supportedResults, first sectionReviewer 2
IRES-TrAPPr is a sensitive and accurate method for high-throughput IRES screening.
The paper provides strong evidence: validation with luciferase assays, high reproducibility (Pearson correlation >0.99), and structure-function analysis confirming known functional regions.
Evidence: IRES-TrAPPr translation efficiency estimates spanned a 1,000-fold range, were highly reproducible, and were validated by independent transfection of 21 IRES-candidate mRNAs.
“IRES-TrAPPr translation efficiency estimates spanned a 1,000-fold range, were highly reproducible ... and were validated by independent transfection of twenty-one IRES-candidate mRNAs cloned upstream of nanoluciferase.”
supportedResults, 'Evaluating viral and cellular IRES candidates nominated by DNA-based assays' sectionReviewers 2, 3
Hundreds of candidate human and viral IRESes from DNA-based screens have negligible IRES activity.
The paper systematically tests 499 viral and 646 cellular IRES candidates from IRESbase and finds only two viral IRESes active; luciferase validation confirms negligible activity.
Evidence: Only two viral IRESes from IRESbase were categorized as 'active' by IRES-TrAPPr; cellular candidates showed negligible activity.
“Surprisingly, only two viral IRESes from IRESbase were categorized as 'active' by IRES-TrAPPr.”
supportedResults, 'Evaluating viral and cellular IRES candidates' sectionReviewer 2
Zika and Dengue virus 5' UTRs have negligible IRES activity even under stress.
IRES-TrAPPr and luciferase assays under various stress conditions (thapsigargin, HRV 2A protease, chemical inhibitors) and in insect cells showed no IRES activity.
Evidence: Zika and Dengue 5' UTRs showed no IRES activity in IRES-TrAPPr, luciferase assays, or in mosquito/fly cells.
“These results show that the Zika and Dengue 5’ UTRs have negligible IRES activity.”
supportedAbstractReviewer 3
IRES-TrAPPr provides a novel, accurate platform for IRES research.
The paper presents extensive validation using known IRESes, structure-function analyses, and comparisons to previous methods, demonstrating the accuracy and utility of IRES-TrAPPr.
Evidence: We validated this new method using luciferase assays and structure-function analyses of established viral IRESes, demonstrating exquisite sensitivity and specificity. Using IRES-TrAPPr, we quantified the activities of IRES elements from hundreds of viruses from a diversity of hosts.
supportedResults, paragraph 5Reviewer 3
The vast majority of short viral IRES elements proposed using DNA-based assays, including MPRAs, are false positives, potentially resulting from artifacts that occur in plasmid reporter systems.
The paper directly tests 499 viral and 646 cellular IRES candidates from DNA-based assays and finds negligible activity for nearly all, strongly supporting the conclusion that many are false positives.
Evidence: Surprisingly, only two viral IRESes from IRESbase were categorized as “active” by IRES-TrAPPr (, ), both of which are Type VI IRESes. Overall, the IRES TE of nearly all of the IRESbase viral IRES candidates we tested was very low compared to CDI translation ().

1 integrity concern flagged (0 high).

Rigor

Eight Dimensions4 pass · 3 warn

11 major claims2 partial9 supported

15 copyedit issues flagged: mostly grammar, consistency, punctuation.

Reproducibility

3 data/code links checked; 1 live.

dataSRALIVEHTTP 200
https://www.ncbi.nlm.nih.gov/sra/
Resolves to SRA (data repository).
codehttp://umi-collapse-singles.pl/UNVERIFIED
Liveness indeterminate — content not checked.
codehttp://umi-collapse-pairs.pl/UNVERIFIED
Liveness indeterminate — content not checked.

Alpha¹ Rigor Action ItemsFinal pass before journal submission

In this post-publication audit, the paper is methodologically sound but has several reporting gaps that an informed reader should weigh when interpreting results. The missing exact p-values and effect sizes limit quantitative scrutiny of comparisons, and the absence of cell line authentication and mycoplasma testing is a reproducibility concern for the cell-based experiments. The incomplete code sharing and missing conflict-of-interest statement are less critical but should be addressed in a correction or data/code update. The core conclusions about IRES-TrAPPr performance and novel IRES elements are supported by the data presented.

1.
HIGHdata code
Deposit all custom analysis scripts (umi-collapse-singles.pl, umi-collapse-pairs.pl, IP_bam_counter_both.pl) in a public repository (e.g., GitHub, Zenodo) and add repository URL and DOI to the Methods section.
Incomplete code sharing limits reproducibility; the current URLs for two of three scripts are insufficient for replication of the full analysis pipeline.
2.
HIGHreporting
Add a conflict-of-interest statement (e.g., 'The authors declare no competing interests') in the Acknowledgments or a dedicated section.
This is standard practice and its absence is a reporting gap that journals may request before acceptance.
3.
HIGHrigor
Report the source (e.g., ATCC) and authentication (e.g., STR profiling) status of HEK293T cells, and state whether mycoplasma testing was performed.
Cell line misidentification and contamination are known reproducibility risks; readers need this information to assess the validity of the cell-based experiments.
4.
HIGHstatistics
Report exact p-values for all statistical comparisons (e.g., Pearson correlations, GC content differences, endothermic vs. ectothermic IRES activity) in the text or supplementary tables.
Qualitative terms like 'approached significance' or 'not statistically significant' do not allow readers to assess the strength of evidence.
5.
HIGHstatistics
Provide effect sizes and confidence intervals for the main group comparisons (e.g., GC content difference between active and inactive IRESes, host-type comparisons).
Effect sizes inform practical significance, and confidence intervals convey precision; their absence weakens quantitative interpretation.
6.
HIGHstatistics
Name the statistical software used for correlation analyses and data visualization (e.g., R version, GraphPad Prism) in the Methods section.
Identification of statistical software is required for reproducibility and is standard reporting practice.
7.
HIGHrigor
State whether statistical test assumptions (e.g., normality, linearity for Pearson correlations) were verified, and if so, how.
The validity of Pearson correlations depends on these assumptions; unverified assumptions may lead to incorrect inferences.
8.
MEDIUMreporting
Include catalog numbers for key reagents: anti-HA magnetic beads (Pierce), Q5 High-Fidelity Polymerase (NEB), Lipofectamine MessengerMAX (Invitrogen), and Twist Bioscience oligo pools.
Catalog numbers enable precise replication and are expected for published methods papers.
9.
MEDIUMcopyedit
Remove the stray text 'pmc 1 preprint 1' and 'May' from the first line of the paper, and ensure the author list starts correctly with 'Gemma E. McManus'.
These appear to be formatting artifacts from the preprint server that would confuse readers and indicate sloppiness.
10.
MEDIUMcopyedit
Fix the inconsistent spelling of 'IRESTrAPPr' (without hyphen) in the Results section title and text to 'IRES-TrAPPr' for consistency.
Inconsistent terminology within a paper suggests editorial oversight and may confuse readers.
11.
MEDIUMcopyedit
Correct the duplicated SRA URL (appears twice) in the data availability statement.
A duplicated URL is a minor formatting error that should be corrected for professional presentation.
12.
MEDIUMcopyedit
Standardize the plural of IRES throughout (IRESes vs. IRESs) — currently used inconsistently — and fix 'bonafide' to 'bona fide'.
Consistent terminology and correct spelling improve readability and adherence to style guidelines.
13.
MEDIUMcopyedit
Add the missing opening parenthesis before the second URL in the sentence about perl scripts (umi-collapse-pairs.pl) in Methods.
This typographical error would break the URL link and reduce clarity.
14.
MEDIUMcopyedit
Define error bars (SD, SEM, or CI) in all figure legends and state which measure of central tendency is shown.
Readers need to know the meaning of error bars to interpret variability in the plots.
15.
MEDIUMreporting
Add a note referencing a relevant reporting guideline (e.g., MIAME for sequencing data or FAIR principles) in the Methods or Data availability section.
Mentioning a reporting guideline demonstrates adherence to community standards and strengthens reproducibility claims.
16.
LOWother
Resolve the internal contradiction flagged by integrity check: clarify whether 93.5% of human and 89.7% of viral IRESbase candidates were included, and if these proportions match the numbers reported (499 viral, 646 cellular).
Readers may question the consistency of these figures; a brief clarification in the Methods would resolve ambiguity.
17.
LOWcopyedit
Remove the redundant 'of' in the phrase 'A- and G- capped of in vitro transcribed RNA pools' in the Immunoprecipitation methods.
Minor grammatical error that does not affect comprehension but indicates lack of proofreading.

Full text (53,946 chars)Analyzed: Jul 6, 2026

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