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Cited by 34RNA and protein synthesis mechanismsRNA Interference and Gene DeliveryRNA Research and Splicing

Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis

Open access
Reshetnikov, Vasiliy, Terenin, Ilya, Shepelkova, Galina, Yeremeev, Vladimir, Kolmykov, Semyon, Nagornykh, Maxim, Kolosova, Elena, Sokolova, Tatiana +10 more·2024·International Journal of Molecular Sciences
DOI 10.3390/ijms25020888PMC PMC10815675
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Abstract

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA—5′ and 3′ untranslated regions (UTRs)—are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein’s expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5′ and 3′UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5′UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5′UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5′UTR also showed higher efficiency (as compared with the 5′UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5′UTRs and 3′UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5′UTRs and 3′UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5′UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

AI snapshot

Written from the paper’s full text.

TL;DR

This study tested how different 5′ and 3′ untranslated region (UTR) sequences affect mRNA translation efficiency and used the best 5′UTR to build a multiepitope mRNA vaccine against *Mycobacterium tuberculosis*. The adenoviral TPL 5′UTR increased luciferase expression in DC2.4 cells and improved T-cell responses and bacterial control in mice compared with the mRNA-1273 5′UTR.

Key findings

  • In DC2.4 cells, 5′UTRs from HBB, HSPA1A, Rabb, H4C2, and TPL increased luciferase bioluminescence at 4 h after transfection compared with the 5′UTRs used in BNT162b2 and mRNA-1273 and compared with C3 [Kruskal–Wallis: H(11, N = 66) = 85.2, p < 0.0001]. In THP1 cells, only HBB showed elevated luciferase at 24 h versus C3 [Kruskal–Wallis: H(11, N = 60) = 31.05, p = 0.001], suggesting cell type-specific effects.
  • 3′UTR effects were detected only in DC2.4 cells at 8, 24, and 72 h [8 h: H(11, N = 66) = 38.37, p < 0.001; 24 h: H(11, N = 66) = 38.49, p < 0.001; 72 h: H(11, N = 66) = 30.49, p = 0.001]. AES-HBB and mtRNR-HBB produced the lowest luciferase levels, and no tested 3′UTR outperformed the mRNA-1273 or BNT162b2 3′UTRs.
  • Predicted mean ribosomal loading (MRL) correlated with bioluminescence in DC2.4 cells at 4 h (r² = 0.43, p = 0.024). TPL had the highest MRL (7.7), versus 5.74 for the mRNA-1273 5′UTR.
  • In mice, the TPL 5′UTR vaccine produced more IFNγ-secreting cells than the mRNA-1273 5′UTR vaccine (13.6 ± 1.9 versus 5.6 ± 0.7). TPL-vaccinated mice also showed delayed-type hypersensitivity and lower lung CFU versus PBS and LNP controls (p < 0.05), whereas the mRNA-1273 5′UTR vaccine differences were not statistically significant.

Methods

  • Twenty-two firefly luciferase mRNA constructs with various 5′ and 3′UTRs were transfected into DC2.4 and THP1 cells; luciferase activity was measured at 4, 8, 24, and 72 h, normalized to NanoLuc, with 4–6 biological replicates.
  • C57BL/6JCit and I/StSnEgYCit mice were immunized twice with 50 μg mRNA-LNP vaccines encoding a multiepitope *M. tuberculosis* antigen, using either TPL or mRNA-1273 as the 5′UTR and mRNA-1273 as the 3′UTR. Outcomes included ELISpot IFNγ responses, delayed-type hypersensitivity, and lung CFU after infection with *M. tuberculosis* H37Rv at 500,000 CFUs per mouse.
  • Bioinformatics included Optimus 5-Prime MRL prediction, RNAFold secondary-structure energy, RBPmap binding-site prediction, and RNA-seq differential expression of RNA-binding protein genes between THP1 and DC2.4 cells. Statistics used Kruskal–Wallis with Dunn’s multiple comparison and correlation analysis.

Limitations

  • The paper states that no significant 3′UTR differences were found, possibly due to assay limitations and the relatively short half-life of luciferase; it notes that definitive UTR conclusions should use direct or indirect target antigen expression and further animal-model research.
  • The paper states that the impact of UTR selection on mRNA stability should be evaluated further, that the MRL predictive model was tested only for 5′UTRs up to 100 bp, and that unmodified uridine was used rather than modified uridine analogs.
  • The text makes evident that the cell-based effects were cell type specific, tested in only two cell lines, and the in vivo comparison tested only TPL versus mRNA-1273 5′UTR with the mRNA-1273 3′UTR, limiting generalization across other UTR combinations, cell types, and vaccine contexts.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/ijms25020888

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

21 of 45 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    UTRs can influence mRNA translation efficiency and resistance to degradation by interacting with RNA-binding proteins and microRNAs.

    “It is known that UTRs are associated with processes of mRNA translation, and by interacting with RNA-binding proteins (RBPs) and microRNAs, UTRs can influence mRNA’s translation efficiency and resistance to degradation [3,4,5,6,7].”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [3] Schlake, 2012, Developing mRNA-vaccine technologies, RNA Biol. source

      The mRNA Design section says UTRs affect translation and stability and that miRNA-binding sites in UTRs can destabilize mRNA. The copy does not explain these effects as arising from interactions with RNA-binding proteins; it mentions UTR-binding factors only in a reference title. No page number is available for these passages. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [4] Lacroix, 2020, Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response, Trends Mol. Med. source
    • cites [5] Xu, S., Yang, K., Li, R., and Zhang, L. (2020). mRNA Vaccine Era-Mechanisms, Drug Platform and Clinical Prospection. Int. J. Mol. Sci., 21. source

      Section 2.2 (page not provided) says UTRs can alter mRNA decay and translation efficiency through interactions with RNA-binding proteins. Section 2.4 mentions microRNA-mediated translation inhibition in its discussion of the poly(A) tail, but does not say that UTRs interact with microRNAs to produce the claimed effects. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [6] Kim, 2022, Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency, Mol. Cell. Toxicol. source

      The “5ʹ- and 3ʹ-UTRs” section says that UTR interactions with RNA-binding proteins can influence mRNA degradation and translation efficiency. It does not mention microRNAs as part of that mechanism, so that portion of the attributed claim is unsupported by our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [7] Kirshina, A., Vasileva, O., Kunyk, D., Seregina, K., Muslimov, A., Ivanov, R., and Reshetnikov, V. (2023). Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA. Biomolecules, 13. source
  • Premisecitation

    There are two experimental studies that demonstrated efficacy of mRNA vaccines against tuberculosis.

    “There are two experimental studies that demonstrated efficacy of the mRNA vaccines against tuberculosis [34,35].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [34] Larsen, S.E., Erasmus, J.H., Reese, V.A., Pecor, T., Archer, J., Kandahar, A., Hsu, F.C., Nicholes, K., Reed, S.G., and Baldwin, S.L. (2023). An RNA-Based Vaccine Platform for Use against Mycobacterium tuberculosis. Vaccines, 11. source
    • cites [35] Xue, 2004, RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection, Infect. Immun. source
  • Premisecitation

    Cap-independent translation plays a major role only during periods of cellular stress, apoptosis, and certain stages of the cell cycle.

    “cap-independent translation plays a major role only during periods of cellular stress, apoptosis, and certain stages of the cell cycle [2].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Pichon, 2012, RNA binding protein/RNA element interactions and the control of translation, Curr. Protein Pept. Sci. source

      The Internal Ribosome Entry Segments section says cap-dependent translation is inhibited during stress, apoptosis, and certain cell-cycle stages, and describes cap-independent translation under stress. It does not establish that cap-independent translation plays a major role only during those periods; the exclusivity and magnitude are unsupported. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The relatively short half-life of luciferase may have an impact on the results, preventing the detection of differences in cumulative transgene expression.

    “The relatively short half-life of luciferase may have an impact on the results [43], preventing the detection of differences in the cumulative transgene expression.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Krishnamachary, 2018, Hypoxia-Induced Reporter Genes with Different Half-Lives, Methods Mol. Biol. source

      The Introduction states that luciferase has a half-life of only 2 hours, and the paper contrasts short-half-life luciferase with long-half-life GFP reporters. It does not state that luciferase's half-life prevented detection of differences in cumulative transgene expression; that proposed effect on the citing study's results is not supported by this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingown data

    AES-EMCV as 3′UTR showed lower bioluminescence values 72 h after transfection of DC2.4 cells compared with mRNA-1273 3′UTR.

    “showed lower bioluminescence values 72 h after the transfection of DC2.4 cells as compared with the mRNA-1273 3′UTR (p < 0.05).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 33
  • May combine two claims. 23
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MtRNR1 is mitochondrial noncoding 12S ribosomal RNA.

    “MtRNR1 is mitochondrial noncoding 12S ribosomal RNA (rRNA) [18].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Chinnery, 2013, Mitochondrial genetics, Br. Med. Bull. source

      The mtDNA > Structure section identifies 12S rRNA as a mitochondrial gene product that is RNA rather than a protein. The Transcription and translation section names MTRNR1 as one of the mtDNA genes contributing to mitoribosomes, but our copy does not explicitly identify MTRNR1 as the 12S rRNA gene. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TPL is a nucleotide sequence in the 5′UTR of adenovirus late mRNAs and has previously been thought to initiate translation of late adenoviral mRNAs in a cap-independent manner.

    “TPL is a nucleotide sequence in the 5′UTR of adenovirus late mRNAs and has previously been thought to initiate translation of late adenoviral mRNAs in a cap-independent manner [15,36].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [15] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source
    • cites [36] Logan, 1984, Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection, Proc. Natl. Acad. Sci. USA source

      The paper’s introductory passage identifies the tripartite leader as a 5′ nontranslated sequence shared by most late adenoviral mRNAs. The Discussion says it likely plays a role in translation initiation, but does not say that initiation is cap-independent; that mechanism is unsupported by this source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Immunization with an mRNA vaccine based on the full-length MPT83 antigen triggered production of specific antituberculosis antibodies.

    “Immunization with an mRNA vaccine based on the full-length MPT83 antigen triggered production of specific antituberculosis antibodies [35].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [35] Xue, 2004, RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection, Infect. Immun. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The direction of the influence of an RBP on the stability and translation efficiency of RNA can depend on the cell type and on the target RNA, as exemplified by PCBP2.

    “Nevertheless, it should be noted that the direction of the influence of an RBP on the stability and translation efficiency of RNA can depend on the cell type and on the target RNA, as exemplified by PCBP2: it can stabilize some mRNAs but promotes the degradation of other mRNAs [25,26,27].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [25] Smirnova, 2019, eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop, RNA source

      The Introduction says poly(rC)-binding proteins can stabilize or destabilize mRNAs, and the Conclusions show that PCBP2’s effects on translation differ by isoform and target RNA. But the paper does not establish that PCBP2 itself stabilizes some mRNAs and promotes degradation of others, or that the direction of its effect reverses by cell type. Its Results and Discussion instead report no significant stability differences among the reporters tested in Huh7 or 293T cells. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [26] Han, 2013, RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3, J. Clin. Investig. source

      The results (section labeled “Conflict of interest”; page unavailable) show that PCBP2 knockdown prolonged FHL3 mRNA half-life in T98G glioma cells, supporting the claim that PCBP2 can promote degradation of a target mRNA. The paper also cites work on stabilization of other mRNAs, but its own experiments do not establish that the direction of PCBP2’s effect depends on cell type. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [27] Ren, 2016, RNA-binding Protein PCBP2 Regulates p73 Expression and p73-dependent Antioxidant Defense, J. Biol. Chem. source

      The Results section reports that loss of PCBP2 shortens p73 mRNA half-life in HCT116 and H1299 cells, supporting PCBP2 as an example of an RBP that stabilizes a target RNA. The paper also describes PCBP2 as regulating the stability and translation of several RNAs, but it does not show PCBP2 promoting degradation of another mRNA or reversing the direction of its effect by cell type. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    There are at least two pathways (cap-dependent and cap-independent) that trigger translation through these elements, and the efficiency of triggering translation via these mechanisms depends on the cellular state.

    “There are at least two pathways (cap-dependent and cap-independent) that trigger translation through these elements, and each pathway requires certain trans-acting proteins; the efficiency of the triggering of translation via these mechanisms depends on the cellular state [2].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [2] Pichon, 2012, RNA binding protein/RNA element interactions and the control of translation, Curr. Protein Pept. Sci. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RBPs can compete for binding to microRNA, thereby having either synergistic or opposing effects on RNA stability.

    “In addition, RBPs can compete for binding to microRNA, thereby having either synergistic or opposing effects on RNA stability [38,39,40].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [38] Orom, 2008, MicroRNA-10a binds the 5’UTR of ribosomal protein mRNAs and enhances their translation, Mol. Cell source

      The Discussion (page not available) speculates that miR-10a competes with an unidentified negative regulator near the 5′TOP motif, which gives limited support for competition involving a microRNA. It does not identify that regulator as an RBP or report synergistic or opposing effects on RNA stability. The Results instead say RP mRNA levels were unaffected and describe predominantly translational regulation. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [39] Peng, 2012, Functional Interactions Between microRNAs and RNA Binding Proteins, MicroRNA source

      The Introduction says RBPs and miRNAs can act synergistically or oppose each other in transcript decay. The section ‘Competition for the Same Binding Position’ describes competition for sites on target mRNAs as antagonistic, not competition for binding to the microRNA itself or competition that produces synergy. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [40] Palanisamy, 2012, Control of cytokine mRNA expression by RNA-binding proteins and microRNAs, J. Dent. Res. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the 5′UTR portions, among the specific sequences that affect translation efficiency, there are the TOP sequence, IRESs, CITEs, uORFs, and protein-binding motifs.

    “In the 5′UTR portions, among the specific sequences that affect translation efficiency, there are the terminal oligopyrimidine tract (TOP) sequence, consisting of 7–14 residues of cytosine and thymine forming a TCT motif; internal ribosome entry sites (IRESs); cap-independent translation enhancers (CITEs); upstream open reading frames (uORFs); and protein-binding motifs [2].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [2] Pichon, 2012, RNA binding protein/RNA element interactions and the control of translation, Curr. Protein Pept. Sci. source

      The Introduction identifies TOP motifs, IRESs and uORFs in the 5′ UTR as elements that influence translation, and the TOP section describes a cytosine followed by a 7–14-residue oligopyrimidine tract. The paper also discusses proteins binding 5′-UTR elements, but the passages do not identify CITEs. The TOP section says a TCT motif is required for transcription of most of these mRNAs; it does not say the oligopyrimidine tract forms that motif. Page numbers were not available in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    AES regulates androgen receptor genes' transcriptional activity and Notch and Wnt signaling, and acts as a tumor suppressor.

    “AES is a distinct member of the family of Groucho/Transducin-like enhancers of split genes; it regulates androgen receptor genes’ transcriptional activity and Notch and Wnt signaling, and acts as a tumor suppressor [19].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Costa, A.M., Pereira-Castro, I., Ricardo, E., Spencer, F., Fisher, S., and da Costa, L.T. (2013). GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos. PLoS ONE, 8. source

      The Introduction (page unavailable) identifies GRG5/AES as a distinct short Groucho family member, and the Results (page unavailable) show that it downregulates Wnt/β-catenin signaling. The paper also cites a study of AES inhibition of androgen receptor-mediated transcription (References, page unavailable). The passages found do not establish that AES regulates Notch signaling or acts as a tumor suppressor; the tumor-suppressor references concern APC, not AES. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The dynamic association of RBPs with mRNA monitors the localization of mRNA, including its movements between actively translating ribosomes, stress granules, and p bodies in a context-dependent manner.

    “Furthermore, the dynamic association of RBPs with mRNA monitors the localization of mRNA, in particular its movements between actively translating ribosomes, stress granules, and p bodies in a context-dependent manner [42].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Bourke, 2023, De-centralizing the Central Dogma: mRNA translation in space and time, Mol. Cell source

      The ‘Ingredients and mechanisms for local translation’ section (page not provided) says that dynamic RBP–mRNA association regulates mRNA transport and translation, and that RBP-dependent localization is context-dependent. It identifies stress granules and P-bodies as associated with translationally repressed mRNA, but does not describe RBP association monitoring mRNA movements between those granules and actively translating ribosomes. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The predictive model (Optimus 5-Prime) was tested only for 5′UTR sequences up to 100 bp in length.

    “It is worth noting that the predictive model in question was tested only for 5′UTR sequences up to 100 bp in length.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] Sample, 2019, Human 5’ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

  • May combine two claims.

    Findingown data

    AES-HBB and mtRNR-HBB as 3′UTR yielded the lowest luciferase levels compared with AES-AES and mtRNR-mtRNR at 8 h, compared with AES-EMCV at 8 and 24 h, and compared with mRNA-1273 3′UTR at 24 and 72 h in DC2.4 cells.

    “Sequences AES-HBB and mtRNR-HBB, when used as the 3′UTR in Luc RNA, yielded the lowest level of luciferase (i) as compared with 3′UTR sequences AES-AES and mtRNR-mtRNR at 8 h after the transfection (p < 0.05), (ii) as compared with 3′UTR sequence AES-EMCV at 8 and 24 h after the transfection (p < 0.05), and (iii) as compared with the 3′UTR from mRNA-1273 at 24 an 72 h after the transfection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice immunized with 5′-mRNA1273-mEpitope-mRNA1273-3′ exhibited a >1.5-fold lower bacterial load compared with control groups, but the differences were statistically insignificant (p > 0.05).

    “Mice immunized with mRNA vaccine 5′-mRNA1273-mEpitope-mRNA1273-3′ also exhibited a >1.5-fold lower bacterial load as compared with the control groups, but the differences were statistically insignificant (p > 0.05).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Zeta potential was in the range −0.22 to −0.37 mV.

    “Zeta potential was in the range −0.22 to −0.37 mV.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The mRNA vaccine containing mRNA-1273 5′UTR produced a numerically larger number of IFNγ-secreting cells compared with control or LNP groups, but the differences were not significant.

    “As for the mRNA vaccine containing the mRNA-1273 5′UTR, the respective experimental group had a numerically larger number of IFNγ-secreting cells compared with the control group (5.6 ± 0.7 vs. 1.3 ± 0.6) or the LNP group (5.6 ± 0.7 vs. 1.0 ± 0.4), but these differences were not significant.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Particle size was 80 to 90 nm.

    “Particle size proved to be 80 to 90 nm”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Only the 3′UTR sequences mtRNR-mtRNR and mRNA-1273, both of which produced the best results, contain a predicted binding site for HNRNPK, which is associated with stabilization of mRNA.

    “Regarding 3′UTRs, only the sequence mtRNR-mtRNR and the mRNA-1273 3′UTR, both of which produced the best results, contain a predicted binding site for an RBP, HNRNPK, which is associated with the stabilization of mRNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The area of target peaks of RNA in capillary electropherograms exceeded 85% of the total RNA.

    “The area of target peaks of RNA in capillary electropherograms exceeded 85% of the total RNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    TPL does not function as an internal ribosome entry site.

    “Furthermore, although it is currently unclear whether the TPL sequence is capable of initiating translation in a cap-independent manner, evidence from our research group suggests that TPL does not function as an internal ribosome entry site (unpublished data).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Three of the top five UTRs (from HBB, Rabb, and H4C2 mRNAs) do not have RBP-binding sites, and TPL and HSPA1A carry binding sites for RBPs whose influence on RNA stability is not clear.

    “Of note, three of the top five UTRs (from HBB, Rabb, and H4C2 mRNAs) do not have RBP-binding sites, and the other two (TPL and HSPA1A) carry binding sites for the RBPs whose influence on RNA stability is not clear.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The polydispersity index was less than 0.15.

    “and the polydispersity index was less than 0.15.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The 5′UTR of rabbit β-globin mRNA is thought to mediate efficient cap-dependent translation and does not contain secondary structures or sequence elements that may negatively affect translation.

    “The 5′UTR of rabbit β-globin mRNA is thought to mediate efficient cap-dependent translation and does not contain any secondary structures or sequence elements that may negatively affect translation [10].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Kozak, 1994, Features in the 5’ non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency, J. Mol. Biol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    3′UTR AU-rich elements can determine the rate of mRNA decay under various conditions.

    “3′UTR AU-rich elements can determine the rate of mRNA decay under various conditions [1,8].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [1] Garneau, 2007, The highways and byways of mRNA decay, Nat. Rev. Mol. Cell Biol. source
    • cites [8] Mayr, 2017, Regulation by 3’-Untranslated Regions, Annu. Rev. Genet. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Sample et al. demonstrated that MRL values range from two to 11; among the highest MRLs, only in the range six-eight was there a high correlation (r2 = 0.77) between predicted and observed values.

    “On the basis of 280,000 different 5′UTR sequences, the Sample et al. [37] study demonstrated that MRL values range from two to 11; however, among the highest MRLs, only in the range six–eight was there a high correlation (r2 = 0.77) between the predicted and observed values.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37] Sample, 2019, Human 5’ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The contribution of cap-independent translation initiation to our results appears negligible because the theoretical amount of capped RNA was ~80% and cap-independent translation plays a major role only during periods of cellular stress, apoptosis, and certain stages of the cell cycle.

    “Nonetheless, the contribution of this alternative mechanism of translation initiation to our results appears to be negligible because the theoretical amount of capped RNA was ~80%, and cap-independent translation plays a major role only during periods of cellular stress, apoptosis, and certain stages of the cell cycle [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Pichon, 2012, RNA binding protein/RNA element interactions and the control of translation, Curr. Protein Pept. Sci. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data

    The 5′-TPL-mEpitope-mRNA1273-3′ vaccine induced stronger adaptive immunity and protection against M. tuberculosis than the mRNA-1273 5′UTR vaccine.

    “Taken together, our results indicate that the 5′-TPL-mEpitope-mRNA1273-3′ vaccine induced stronger adaptive immunity and protection against M. tuberculosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Use of TPL as a 5′UTR compared with the 5′UTR from mRNA-1273 evoked more pronounced protective adaptive immune responses in mice, leading to higher preclinical efficacy of mRNA vaccines against tuberculosis.

    “Our study is the first one to compare mRNA vaccines with various 5′UTRs; we have shown that use of TPL as a 5′UTR compared with the 5′UTR from mRNA-1273 evoked more pronounced protective adaptive immune responses in mice leading to higher preclinical efficacy of mRNA vaccines against tuberculosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    mRNA therapeutics require optimization of mRNA sequences because their effectiveness primarily depends on the rate of degradation and the rate of translation initiation.

    “Nevertheless, mRNA therapeutics require the optimization of mRNA sequences because their effectiveness primarily depends on the rate of degradation (and the rate of translation initiation) of mRNA [1,2].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Garneau, 2007, The highways and byways of mRNA decay, Nat. Rev. Mol. Cell Biol. source
    • cites [2] Pichon, 2012, RNA binding protein/RNA element interactions and the control of translation, Curr. Protein Pept. Sci. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    C57BL/6Cit and I/StSnEgYCit mice are extremely susceptible to tuberculosis.

    “C57BL/6Cit and I/StSnEgYCit mice, which are extremely susceptible to tuberculosis [33].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] Radaeva, 2005, Direct comparison of low-dose and Cornell-like models of chronic and reactivation tuberculosis in genetically susceptible I/St and resistant B6 mice, Tuberculosis source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The transfection procedures and bioluminescence assessment for each construct were performed in 4-6 biological replicates, with 3 technical repetitions for each.

    “The transfection procedures and bioluminescence assessment for each construct were performed in 4–6 biological replicates (with 3 technical repetitions for each).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Adenovirus TPL can enhance protein expression by enhancing translation initiation.

    “Some regulatory elements, such as adenovirus TPL (tripartite leader), can enhance protein expression by enhancing translation initiation [15].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Globin gene sequences are often used to ensure high translation efficiency.

    “Globin gene sequences are often used to ensure high translation efficiency.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [9] Babendure, 2006, Control of mammalian translation by mRNA structure near caps, RNA source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [10] Kozak, 1994, Features in the 5’ non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency, J. Mol. Biol. source
    • cites [11] Annweiler, 1991, A strategy for efficient in vitro translation of cDNAs using the rabbit beta-globin leader sequence, Nucleic Acids Res. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Another mRNA vaccine contains four full-length tuberculosis antigens fused with a synthetic TLR4 agonist.

    “Another mRNA vaccine contains four full-length tuberculosis antigens fused with a synthetic TLR4 agonist [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Larsen, S.E., Erasmus, J.H., Reese, V.A., Pecor, T., Archer, J., Kandahar, A., Hsu, F.C., Nicholes, K., Reed, S.G., and Baldwin, S.L. (2023). An RNA-Based Vaccine Platform for Use against Mycobacterium tuberculosis. Vaccines, 11. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationnone given

    The function of PABPC3 and RBM22 is related to ensuring the stability of the poly(A) tail or regulation of alternative splicing and plays a negligible role in the translation of heterologous RNAs.

    “however, the function of these RBPs is related to either ensuring the stability of the poly(A) tail or regulation of alternative splicing and plays a negligible role in the translation of heterologous RNAs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RBPs monitor mRNA stability and decay and control the correct translation of mRNA.

    “RBPs monitor mRNA stability and decay and control the correct translation of mRNA.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Bourke, 2023, De-centralizing the Central Dogma: mRNA translation in space and time, Mol. Cell source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All obtained data were not normally distributed.

    “All obtained data were not normally distributed, and therefore we chose nonparametric analysis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The transport function of RBPs can be mediated either by their direct binding to motor proteins or indirectly through motor protein cargo adaptors.

    “The transport function of RBPs can be mediated either by their direct binding to motor proteins or, indirectly, through motor protein cargo adaptors [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Bourke, 2023, De-centralizing the Central Dogma: mRNA translation in space and time, Mol. Cell source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Bioluminescence data from firefly luciferase were normalized to the bioluminescence of NanoLuc luciferase, which served as an internal control.

    “Bioluminescence data from firefly luciferase were normalized to the bioluminescence of NanoLuc luciferase, which served as an internal control.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The desired length and homogeneity of the synthesized RNA molecules were verified by capillary electrophoresis.

    “The desired length and homogeneity of the synthesized RNA molecules were verified by capillary electrophoresis on a TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Effects of many RBPs on translation are yet to be identified.

    “Nonetheless, effects of many RBPs on translation are yet to be identified [41].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Gerstberger, 2014, A census of human RNA-binding proteins, Nat. Rev. Genet. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Orlandini von Niessen et al. showed that 3′UTRs containing mtRNR1 and AES promote the expression of the upstream gene.

    “They showed that 3′UTRs containing mtRNR1 and AES promote the expression of the upstream gene.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Poleganov, 2019, Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3’ UTRs Identified by Cellular Library Screening, Mol. Ther. J. Am. Soc. Gene Ther. source

  • May combine two claims.

    Interpretationreasoning

    A direct relation between the presence of an RBP landing site and RNA stability was difficult to discern because some RBPs stabilize RNA, some destabilize it, and others regulate RNA stability in a context-dependent manner.

    “Nonetheless, a direct relation between the presence of a landing site and the RNA stability was difficult to discern because some RBPs stabilize RNA, some destabilize it, and other RBPs can regulate RNA stability in a context-dependent manner, as is the case for PCBP2 (Table 2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Regulatory sequences of constitutively expressed genes are often used to enhance the translation efficiency of heterologous mRNAs.

    “To sum up the data on natural 5′UTRs, one can conclude that regulatory sequences of constitutively expressed genes are often used to enhance the translation efficiency of heterologous mRNAs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The effect of HSP70 5′UTR has been observed in several cell lines, indicating that this effect is not cell type specific.

    “The effect of HSP70 5′UTR has been observed in several cell lines [14], indicating that this effect is not cell type specific.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Vivinus, 2001, An element within the 5’ untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation, Eur. J. Biochem. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TPL contains an internal ribosome entry site enabling it to recruit a ribosome independently of interactions with the mRNA 5′ 7-methyl G cap.

    “TPL contains an internal ribosome entry site enabling it to recruit a ribosome independently of interactions with the mRNA 5′ 7-methyl G cap.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [15] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source
    • cites [36] Logan, 1984, Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection, Proc. Natl. Acad. Sci. USA source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Cells without stimulation or stimulated with concanavalin A served as negative and positive controls in the ELISpot procedure.

    “Cells without the stimulation or stimulated with concanavalin A (Sigma-Aldrich, Darmstadt, Germany) at a dose of 5 μg/mL served, respectively, as negative and positive controls in the ELISpot procedure.”
    Quote in the paperNot a substantial claim
45 of 45

  • The paper relies on this without support.

    The 3′UTR is primarily responsible for RNA stability in the cell and prevents RNA degradation mediated by binding to microRNA.

    “Because the 3′UTR is primarily responsible for RNA stability in the cell and prevents the RNA degradation mediated by binding to microRNA, our findings implied that 3′UTR sequences AES-HBB and mtRNR-HBB within mRNA lead to a decrease in its stability.”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In mRNA vaccines, use of different 5′ UTR sequences can substantially enhance immunological properties, including increased antibody titers and improved T-cell responses.

    “In the setting of mRNA vaccines, the use of different 5′ UTR sequences can substantially enhance immunological properties, including increased antibody titers and improved T-cell responses [18,19].”

    From Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity (2026) · cites this paper as [18]

    Results §2.5 reports that a tuberculosis mRNA vaccine with the TPL 5′ UTR produced more than twice as many IFNγ-secreting cells in immunized mice as one with the mRNA-1273 5′ UTR, supporting the T-cell-response portion. The paper does not report increased antibody titers from changing 5′ UTRs; the antibody finding mentioned in Discussion §3 concerns a different study. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Kruskal-Wallis statistic exceeds its maximum for 66 observations

    Major

    For N = 66, the Kruskal-Wallis H statistic cannot exceed N − 1 = 65, including with a tie correction. The reported H of 85.2 is therefore impossible for the stated sample size.

    Reported 85.2 · recomputed 65

  • THP1 5′UTR effect is both reported and denied

    Major

    The abstract says no effect of the tested UTRs was detectable in THP1 cells, but the results report a significant 5′UTR effect there at 24 hours.

  • Discussion denies reported 3′UTR differences

    Major

    The discussion says there were no significant differences among tested 3′UTRs, whereas the results report a significant 3′UTR effect in DC2.4 cells at three time points.

3 of 3

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Authors
Reshetnikov, V., Terenin, I., Shepelkova, G., Yeremeev, V., Kolmykov, S., Nagornykh, M., Kolosova, E., Sokolova, T., Zaborova, O., Kukushkin, I., Kazakova, A., Kunyk, D., Kirshina, A., Vasileva, O., Seregina, K., Pateev, I., Kolpakov, F., & Ivanov, R.
Affiliations
  • Russian Academy of Sciences (RU)
  • Lomonosov Moscow State University (RU)
  • Central Tuberculosis Research Institute, 107564 Moscow, Russia
  • Central Scientific Research Institute of Tuberculosis Russian Academy of Medical Sciences (RU)
  • Sirius University of Science and Technology (RU)
  • Translational Medicine Research Center, Sirius University of Science and Technology, 354340 Sochi, Russia
Corresponding author
Reshetnikov, Vasiliy
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2024-01-10
Volume
25
Issue
2
Pages
888
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms25020888
PMC
PMC10815675
OpenAlex
W4390806630
Cited by
33
References
54
Citations by year
2026: 15 · 2025: 13 · 2024: 5
FWCI
4.27
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Ministry of Science and Higher Education of the Russian Federation (075-10-2021-113)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
mRNA vaccines, tuberculosis, untranslated regions, 5' untranslated region, 3' untranslated region, translation efficiency, firefly luciferase, T cell response, mRNA-1273, BNT162b2, DC2.4 cells, THP-1 cells
MeSH terms
mRNA Vaccines, BNT162 Vaccine, Animals, Blood Group Antigens, Humans, Mice, Inbred C57BL, RNA, Messenger, Tuberculosis, 5' Untranslated Regions, 3' Untranslated Regions, Mice, Pandemics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-05.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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