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Cited by 27RNA and protein synthesis mechanismsRNA Research and SplicingRNA Interference and Gene Delivery

Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA

Open access
Kirshina, Anna, Vasileva, Olga, Kunyk, Dmitry, Seregina, Kristina, Muslimov, Albert, Ivanov, Roman, Reshetnikov, Vasiliy·2023·Biomolecules
DOI 10.3390/biom13111677PMC PMC10669451
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Abstract

mRNA-based therapeutics have been found to be a promising treatment strategy in immunotherapy, gene therapy, and cancer treatments. Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein’s expression, which is determined by various cis- and trans-regulatory elements of the mRNA. Sequences of 5′ and 3′ untranslated regions (UTRs) are responsible for translational efficiency and stability of mRNA. An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein’s expression. Using both literature data and previously obtained experimental data, we chose six sequences of 5′UTRs (adenoviral tripartite leader [TPL], HBB, rabbit β-globin [Rabb], H4C2, Moderna, and Neo2) and five sequences of 3′UTRs (mtRNR-EMCV, mtRNR-AES, mtRNR-mtRNR, BioNTech, and Moderna). By combining them, we constructed 30 in vitro transcribed RNAs encoding firefly luciferase with various combinations of 5′- and 3′UTRs, and the resultant bioluminescence was assessed in the DC2.4 cell line at 4, 8, 24, and 72 h after transfection. The cellular data enabled us to identify the best seven combinations of 5′- and 3′UTRs, whose translational efficiency was then assessed in BALB/c mice. Two combinations of 5′- and 3′UTRs (5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech) led to the most pronounced increase in the luciferase amount in the in vivo experiment in mice. Subsequent analysis of the stability of the mRNA indicated that the increase in luciferase expression is explained primarily by the efficiency of translation, not by the number of RNA molecules. Altogether, these findings suggest that 5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR- EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.

AI snapshot

Written from the paper’s full text.

TL;DR

Combinations of 5′ and 3′ UTRs strongly influence mRNA translation, and two combinations—5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech—gave the highest luciferase expression in DC2.4 cells and BALB/c mice. The increased expression was driven mainly by translational efficiency rather than by higher mRNA copy number.

Key findings

  • In DC2.4 cells, UTR sequence affected luciferase bioluminescence at 4, 8, 24, and 72 h after transfection (one-way ANOVA, all p < 0.001). 5′TPL-3′mtRNR-AES and 5′Rabb-3′mtRNR-AES produced higher bioluminescence than 5′Moderna-3′Moderna and 5′Biontech-3′Biontech at 8, 24, and 72 h; 5′TPL-3′Biontech, 5′H4C2-3′mtRNR-mtRNR, and 5′Neo2-3′mtRNR-mtRNR enhanced expression at 8 and/or 24 h, but differences disappeared by 72 h.
  • In BALB/c mice, UTR combinations affected bioluminescence at 4, 8, 24, and 48 h (Kruskal–Wallis: 4 h p = 0.0056; 8 h p = 0.064; 24 h p = 0.038; 48 h p = 0.024), with no significant differences at 72 h (p = 0.71). Only 5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech significantly enhanced bioluminescence versus 5′Moderna-3′Moderna at 8, 24, and 48 h.
  • At 24 h after injection, FFLuc mRNA copy numbers did not differ significantly among groups: 5′Rabb-3′mtRNR-EMCV had 53.4 ± 45.6 million copies, 5′TPL-3′Biontech had 29.7 ± 12.4 million, and 5′Moderna-3′Moderna had 20.8 ± 4.1 million (1.4–2.5-fold differences). By 72 h, all groups showed an approximately 2- to 4-fold decrease and the intergroup differences disappeared.
  • The two best combinations gave approximately 6-fold higher bioluminescence per RNA molecule at 24 h, suggesting the effect was mediated primarily by enhanced translational efficiency and to a lesser extent by increased RNA stability.

Methods

  • The authors constructed 30 in vitro–transcribed mRNAs encoding firefly luciferase with combinations of six 5′UTRs (TPL, HBB, rabbit β-globin [Rabb], H4C2, Moderna, Neo2) and five 3′UTRs (mtRNR-EMCV, mtRNR-AES, mtRNR-mtRNR, BioNTech, Moderna), plus a BioNTech-reference mRNA (31 total). These were transfected into mouse DC2.4 dendritic cells, and bioluminescence was measured at 4, 8, 24, and 72 h with NanoLuc mRNA as an internal normalization control.
  • The best seven combinations from the cellular data were encapsulated into lipid nanoparticles and injected intramuscularly at 5 µg into 64 adult male BALB/c mice randomized into eight groups (8 mice/group). Bioluminescence was assessed at 4, 8, 24, 48, or 72 h, and FFLuc mRNA copy number at the injection site was measured by droplet digital PCR at 24 and 72 h.
  • Cell-culture data were analyzed by ANOVA with Bonferroni post hoc tests; in vivo data were analyzed by Kruskal–Wallis tests with Dunn’s multiple-comparison post hoc tests, with significance at p < 0.05.

Limitations

  • The paper does not state limitations explicitly. Evident from the text: the in vitro work used only the DC2.4 dendritic cell line, and the in vivo work used only male BALB/c mice, a single intramuscular 5 µg mRNA-LNP dose, and one LNP formulation, so generalization to other cell types, species, doses, or delivery systems is not established.
  • The in vivo 8 h Kruskal–Wallis result was p = 0.064, not below the stated p < 0.05 threshold, and no significant differences were found at 72 h; mRNA copy-number differences at 24 h were also not statistically significant.
  • The mechanistic interpretation is largely speculative: the study measured bioluminescence and mRNA copy number but did not directly assay ribosome loading, translation initiation, or UTR–protein interactions.

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

Integrity signals

Registry

Crossref · 10.3390/biom13111677

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

16 of 44 cited sources checked · 6 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    The authors of ref. [46] also demonstrated that MRL inversely correlates with minimal free energy values.

    “It should be pointed out that the authors of ref. [46] also demonstrated that MRL inversely correlates with minimal free energy values.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

      The Results section (‘Evaluation of mRNA containing Ψ and m1Ψ’) reports a positive, not inverse, correlation between mean ribosome load and predicted minimum free energy for U, Ψ, and m1Ψ. The supplied passage does not give a page number. (LLM, conf=0.92)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    Neo-2 has previously manifested high translational efficiency.

    “In addition, we used synthetic sequence Neo-2, which has previously manifested high translational efficiency too [35].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [35] Cao, 2021, High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies, Nat. Commun. source

      The Results section ('Synthetic 5′ UTRs enhance protein expression levels in non-viral DNA therapies'; page unavailable) reports that NeoUTR2 increased GFP abundance and VEGF production. It does not report a measured translational efficiency for NeoUTR2; the paper distinguishes translational efficiency from protein expression, so the specific efficiency claim is not established. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    eIF4A can bind to mRNA immediately before translation initiation and unwind such mRNA structures, thereby clearing the path for scanning.

    “initiation factor 4A (eIF4A) can bind to mRNA immediately before translation initiation and unwind such mRNA structures, thereby clearing the path for scanning [9].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] Kumari, 2007, An RNA G-quadruplex in the 5′ UTR of the NRAS proto-oncogene modulates translation, Nat. Chem. Biol. source

      The paper’s Abstract reports that an NRAS 5′-UTR G-quadruplex inhibits translation, but says its inhibitory mechanism remains to be elucidated. Searches for eIF4A, helicase activity, and scanning found no passage stating that eIF4A binds and unwinds mRNA structures before initiation. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
  • Premisecitation

    Secondary structures of 5′UTRs can function as major regulatory tools in relation to translation.

    “Secondary structures of 5′ UTRs can function as major regulatory tools in relation to translation [7].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [7] Araujo, 2012, Before It Gets Started: Regulating Translation at the 5′ UTR, Comp. Funct. Genom. source
  • Premisecitation

    On the basis of 280,000 different 5′UTR sequences, the authors of that paper demonstrated that MRL values range from 2 to 11.

    “On the basis of 280,000 different 5′UTR sequences, the authors of that paper demonstrated that MRL values range from 2 to 11.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

      Online Methods (“Sequence Processing”) says the authors selected 280,000 distinct 5′ UTR sequences for the eGFP library and calculated mean ribosome load (MRL) for them. The Results (“Modeling 5′ UTRs and ribosome loading”) also identifies the 280,000-member library, but the passages found do not establish that its MRL values ranged from 2 to 11. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 20
  • May combine two claims. 24
  • The quote only partly states this claim. 1

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

    Premisecitation

    The 5′UTR and 3′UTR of p53 mRNA contain complementary regions that bind translation factor RPL26, which mediates translation enhancement in response to DNA damage.

    “Although a 5′UTR and 3′UTR do not interact directly during translation, it has been demonstrated by means of p53 mRNA as an example that the 5′UTR and 3′UTR contain complementary regions that bind translation factor RPL26, which mediates translation enhancement in response to DNA damage [40].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] Chen, 2010, 5′-3′-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage, Genes Dev. source

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

    Premisecitation

    Sequences of human or rabbit α- and β-globin or of rabbit β-globin have been used to improve translational efficiency of heterologous mRNAs.

    “In particular, sequences of human or rabbit α- and β-globin or of rabbit β-globin have been used to improve translational efficiency of heterologous mRNAs [36,37,38].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [36] Babendure, 2006, Control of mammalian translation by mRNA structure near caps, RNA source

      The ‘Translation efficiency with natural 5′ UTRs’ section reports cloning α- and β-globin 5′ UTRs into a GFP reporter and finding greater translation efficiency than with the designed hairpin set. This supports the use of globin sequences with a heterologous mRNA, but the paper does not identify those globin sequences as human or rabbit or establish every species-specific example in the citing sentence. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [37] 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 [38] 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

    TPL contains an IRES that allows it to recruit a ribosome independently of interactions with the 5′ 7-methyl G cap of mRNA.

    “This research [43,44] indicates that TPL contains an IRES due to which TPL is capable of recruiting a ribosome, independently of interactions with the 5′ 7-methyl G cap of mRNA.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [43] Logan, 1984, Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection, Proc. Natl. Acad. Sci. USA source

      The Results report that the tripartite leader enhances translation late after infection, and the Discussion suggests it may act during initiation. But the Discussion says its point of action is unclear; our copy does not establish that it contains an IRES or recruits ribosomes independently of the 5′ cap (page numbers unavailable). (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [44] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source

  • May combine two claims.

    Premisecitation

    Higher GC content and highly negative folding minimal free energy (ΔG) of 5′UTRs correlate with inhibition of translation.

    “In addition to greater length, the TPL sequence has a higher GC content and highly negative folding minimal free energy (ΔG), which correlates with inhibition of translation [4,7,45,46] (Table 1).”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [4] Leppek, 2018, Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them, Nat. Rev. Mol. Cell Biol. source
    • cites [7] Araujo, 2012, Before It Gets Started: Regulating Translation at the 5′ UTR, Comp. Funct. Genom. source
    • cites [45] Pelletier, 1985, Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency, Cell source
    • cites [46] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

      In Results, “MPRA design and validation” (page unavailable), the paper reports that strong RNA secondary structures have a negative effect on translation and that comparing 5′ UTR minimum free energy with ribosome load captured this repressive effect. It does not establish a separate correlation between higher GC content and translation inhibition; the TPL sequence measurements are the citing authors’ own observations. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Translation from the EMCV IRES is strengthened by the pyrimidine tract-binding protein (PTB).

    “Moreover, translation from the EMCV IRES is strengthened by the pyrimidine tract–binding protein (PTB) [52].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [52] Chamond, 2014, 40S recruitment in the absence of eIF4G/4A by EMCV IRES refines the model for translation initiation on the archetype of Type II IRESs, Nucleic Acids Res. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Optimus 5-Prime is a method for predicting MRL of a transcript through the use of convolutional neural networks.

    “The latter is a method for predicting MRL of a transcript through the use of convolutional neural networks.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

  • May combine two claims.

    Premisecitation

    TPL as the 5′UTR had the highest MRL (7.7), whereas MRL of the other tested UTRs was within 5.7-6.7.

    “TPL as the 5′UTR had the highest MRL (7.7), whereas MRL of the other tested UTRs was within 5.7–6.7.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source

      The Results (MPRA design and validation; page unavailable) describe how mean ribosome load was measured, but do not identify TPL as a tested 5′ UTR. Searches of our copy for TPL, 7.7, and the 5.7–6.7 range found no passage reporting the claimed comparison. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Premisecitation

    IRE is a small region of a loop in a 5′UTR near the cap of a subpopulation of mRNAs involved in iron homeostasis.

    “A classic example is the iron-responsive element (IRE), which is a small region of a loop in a 5′UTR near the cap of a subpopulation of mRNAs involved in iron homeostasis [10].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [10] Banai, 2002, Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR, Cell source

      The Results passage concerns the 5′-terminal region of IFN-γ mRNA and its activation of PKR, not an iron-responsive element. Searches for IRE, iron-responsive elements, ferritin, and iron homeostasis found no passage in our copy describing the claimed location or role of an IRE. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

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

    Findingown data

    The LNP particles had a size of 80-90 nm and a polydispersity index less than 0.15.

    “Particle size proved to be from 80 to 90 nm, and the polydispersity index was less than 0.15.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    The approximately 6-fold higher bioluminescence per RNA molecule for 5′TPL-3′Biontech and 5′Rabb-3′mtRNR-EMCV was undetectable at 72 h after injection in BALB/c mice.

    “These differences, however, were undetectable at 72 h after the injection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In DC2.4 cells, FFLuc mRNA with UTR combination 5′Rabb-3′mtRNR-EMCV resulted in 18% higher luciferase at 72 h after transfection compared with control, but the difference was not statistically significant (p = 0.31).

    “In addition, combination 5′Rabb-3′mtRNR-EMCV was selected for the next stage of testing; this combination within mRNA resulted in luciferase upregulation by 18% (p = 0.31) at 72 h after transfection of DC2.4 cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The TPL sequence has a higher GC content and a more negative folding minimal free energy (ΔG) than the reference 5′UTRs (Table 1).

    “In addition to greater length, the TPL sequence has a higher GC content and highly negative folding minimal free energy (ΔG), which correlates with inhibition of translation [4,7,45,46] (Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In DC2.4 cells, FFLuc mRNA with UTR combination 5′H4C2-3′Biontech did not significantly increase translational efficiency at early time points but caused luciferase overexpression at 72 h after transfection compared with mRNA containing 5′Biontech-3′Biontech (p < 0.05).

    “On the contrary, UTR combination 5′H4C2-3′Biontech within mRNA did not lead to a significant increase in translational efficiency of mRNA at the early time points but caused luciferase overexpression at 72 h after the transfection (p < 0.05) as compared to the mRNA containing 5′Biontech-3′Biontech.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In DC2.4 cells, FFLuc mRNA carrying UTR combinations 5′TPL-3′mtRNR-AES or 5′Rabb-3′mtRNR-AES produced higher bioluminescence than mRNA carrying 5′Moderna-3′Moderna or 5′Biontech-3′Biontech at 8, 24, and 72 h after transfection.

    “UTR combinations 5′TPL-3′mtRNR-AES and 5′Rabb-3′mtRNR-AES within FFLuc mRNA resulted in higher levels of bioluminescence as compared to 5′Moderna-3′Moderna and 5′Biontech-3′Biontech at three time points (8, 24, and 72 h after transfection).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 24 h after administration in BALB/c mice, injection sites of mRNA carrying 5′Rabb-3′mtRNR-EMCV had 53.4 ± 45.6 million copies and those carrying 5′TPL-3′Biontech had 29.7 ± 12.4 million copies, which was 1.4-2.5 times greater than mRNA containing 5′Moderna-3′Moderna (20.8 ± 4.1 million copies).

    “Nevertheless, at the site of injection of mRNA carrying 5′Rabb-3′mtRNR-EMCV, there were 53.4 ± 45.6 million copies (mean ± SEM), and at the site of injection of mRNA containing 5′TPL-3′Biontech, there were 29.7 ± 12.4 million copies, which was 1.4–2.5 times greater relative to mRNA containing 5′Moderna-3′Moderna (20.8 ± 4.1 million copies).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The reference 5′UTR sequences from mRNA-1273 (Moderna) and BNT162b2 (BioNTech) used in this work had lengths of 57 and 54 nucleotides, respectively, and did not contain strong secondary structures (Table 1).

    “Similarly, in our work, reference types of 5′UTR sequences from vaccines mRNA-1273 and BNT162b2 (Moderna and BioNTech) had a length of 57 nucleotides (Moderna, Cambridge, MA, USA; synthetic sequence) and 54 nucleotides (BioNTech, Mainz, Germany; 97% homologous to the 5′UTR of the human α-globin gene) and did not contain strong secondary structures (Table 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.

    “Altogether, these findings suggest that 5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR- EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein's expression.

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

    Premisecitation

    Longer 3′UTRs correlate with lower expression levels.

    “Longer 3′UTRs correlate with lower expression levels [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Sandberg, 2008, Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites, Science source

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

    Study design and conductown data

    The synthesized RNA molecules were verified to have the desired length and homogeneity by capillary electrophoresis on a TapeStation instrument.

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

  • May combine two claims.

    Premisecitation

    mtRNR1, AES, and EMCV individually have previously been found to influence mRNA stability.

    “As 3′UTRs, various combinations of mtRNR1, AES, and EMCV were chosen, which individually have previously also been found to influence mRNA stability [34,39].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [34] 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
    • cites [39] Paek, 2015, Translation initiation mediated by RNA looping, Proc. Natl. Acad. Sci. USA 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

    Bioluminescence of experimental FFLuc mRNA was normalized to NanoLuc luciferase mRNA, which served as the internal control.

    “mRNA of NanoLuc luciferase served as the mRNA for normalization of data (the internal control).”
    • Also stated in: § 2.5. Bioluminescence Detection · plain
      “To obtain the final value, the bioluminescence of the experimental FFLuc mRNA was normalized to the bioluminescence of NanoLuc mRNA.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In comparison with recombinant-protein drugs, the development of mRNA-based therapeutics is faster and more flexible owing to the transcription of mRNA in a cell-free in vitro system.

    “In comparison with recombinant-protein drugs, the development of mRNA-based therapeutics is faster and more flexible owing to the transcription of mRNA in a cell-free in vitro system [2].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Weng, Y., Li, C., Yang, T., Hu, B., Zhang, M., Guo, S., Xiao, H., Liang, X.J., and Huang, Y. (2020). The challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv., 40. source

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

    Study design and conductnone given

    The mice were subdivided into eight groups via randomization, with 8 mice per group.

    “The animals were subdivided into eight groups via randomization, at 8 mice per group.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A 3′UTR participates in alternative cleavage and polyadenylation, translation, and localization of mRNA, affecting mRNA stability.

    “A 3′UTR participates in such processes as alternative cleavage and polyadenylation, translation, and localization of mRNA [19], affecting mRNA stability [20].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Martin, 2009, mRNA localization: Gene expression in the spatial dimension, Cell source

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

    • cites [20] Chen, 1995, AU-rich elements: Characterization and importance in mRNA degradation, Trends Biochem. Sci. source

  • May combine two claims.

    Premisecitation

    Low stability of RNA in the cell causes premature degradation of RNA, low translational efficiency, and a decrease in the level and duration of expression of the target protein.

    “Low stability of RNA in the cell causes premature degradation of RNA, low translational efficiency, and a decrease in the level and duration of expression of the target protein [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Weng, Y., Li, C., Yang, T., Hu, B., Zhang, M., Guo, S., Xiao, H., Liang, X.J., and Huang, Y. (2020). The challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv., 40. source

  • May combine two claims.

    Premisecitation

    Sequences of 5′ and 3′ untranslated regions (UTRs) play a key role in the stability of mRNA molecules and in their translational efficiency.

    “Sequences of 5′ and 3′ untranslated regions (UTRs) are responsible for translational efficiency and stability of mRNA.”
    • Also stated in: § 1. Introduction · plain
      “One of the key roles in the stability of mRNA molecules and in their translational efficiency is played by regulatory sequences of untranslated regions (5′UTR and 3′UTR) [3,4].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [3] Matoulkova, 2012, The role of the 3′ untranslated region in post-transcriptional regulation of protein expression in mammalian cells, RNA Biol. source

      Not checked in the free run.

    • cites [4] Leppek, 2018, Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them, Nat. Rev. Mol. Cell Biol. source

      Not checked in the free run.

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

    Premisecitation

    The sequence of the encephalomyocarditis virus (EMCV) IRES comprises strong secondary structures and has been used to enhance the expression of heterologous RNAs.

    “The sequence of the encephalomyocarditis virus (EMCV) IRES comprises strong secondary structures and has been used to enhance the expression of heterologous RNAs [39].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Paek, 2015, Translation initiation mediated by RNA looping, 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), S4 (based). May combine two claims.

    Interpretationnone given

    Although the 5′UTR and 3′UTR are currently viewed as discrete regulatory units that do not interact directly with each other, accumulating evidence points to their indirect influence on each other.

    “Although the 5′UTR and 3′UTR are currently viewed as discrete regulatory units that do not interact directly with each other, accumulating evidence points to their indirect influence on each other.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    In prior studies, the 5′UTR sequence of TPL consisted of 245 nucleotides and had a complex secondary structure.

    “The 5′UTR sequence of TPL consisted of 245 nucleotides in those studies and had a complex secondary structure.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [43] 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.

    • cites [44] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source

  • May combine two claims.

    Interpretationreasoning

    The EMCV sequence within the mRNA carrying 5′-Rabb-3′mtRNR-EMCV may play a role not only in stability but also in translational efficiency of the mRNA.

    “On the basis of these suppositions, we can conclude that the EMCV sequence within the mRNA carrying 5′-Rabb-3′mtRNR-EMCV may play a role not only in stability but also in translational efficiency of the mRNA.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: For cap-independent translation initiation involving the EMCV IRES, the binding of the J-K domain to eIF4G is needed for the recruitment of preinitiation complex 43S.
    • The authors state this as an assumption: Translation enhancement via ribosomes recruited downstream of a gene may proceed without transfer of a ribosome to the 5′ end, because the 43S recruited to the 3′UTR can find the start codon in a 5′-end-independent manner via circularization of an intermediate RNA region separating the 5′UTR and 3′UTR.

  • May combine two claims.

    Premisecitation

    Some mRNAs with a long 5′UTR and high GC content are actively translated via a cap-dependent mechanism; the 5′UTR of LINE1 mRNA (900-nt, 60% GC content) is an example.

    “By contrast, there are mRNAs with a long 5′UTR that has a high GC content, and yet these mRNAs are actively translated via a cap-dependent mechanism; the 5′UTR of LINE1 mRNA is an example (900-nt 5′UTR with a 60% GC content) [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Beaudoin, 2010, 5′-UTR G-quadruplex structures acting as translational repressors, Nucleic Acids Res. 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

    The IRE in mRNA encoding ferritin or ferroportin associates with IRP1 or IRP2 under low-iron conditions.

    “The IRE in mRNA encoding either ferritin (iron storage protein) or ferroportin (iron transporter) associates with iron regulatory protein 1 or 2 (IRP1 or IRP2) under low-iron conditions.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Banai, 2002, Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR, 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).

    Premisecitation

    If IRPs bind to a 3′UTR, then they enhance the stability of the mRNA.

    “On the other hand, if IRPs bind to a 3′UTR, then they enhance the stability of the mRNA [49].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Kuhn, 2015, Iron regulatory proteins and their role in controlling iron metabolism, Met. Integr. Biometal Sci. source

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

    Interpretationown data

    The observed 1.4-2.5-fold increase in FFLuc mRNA copy number at the injection site after 24 h for 5′TPL-3′Biontech or 5′Rabb-3′mtRNR-EMCV suggests some improvement of RNA stability.

    “On the other hand, the observed increase in the copy number of mRNA carrying 5′TPL-3′Biontech or 5′Rabb-3′mtRNR-EMCV by 1.4–2.5-fold at the injection site after 24 h allowed us to hypothesize that there was some improvement of RNA stability.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The number of mRNA copies at the injection site reflects the stability of the mRNA.

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

    Premisecitation

    The adenovirus tripartite leader (TPL) functions as an enhancer for translation of viral late-gene mRNAs and is thought to initiate translation of late adenoviral mRNAs in a cap-independent manner.

    “The tripartite leader (TPL) of the adenovirus is known to function as an enhancer for translation of viral late-gene mRNAs and is thought to have an ability to initiate translation of late adenoviral mRNAs in a cap-independent manner [43,44].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [43] 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.

    • cites [44] Kaufman, 1985, Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors, Proc. Natl. Acad. Sci. USA source

  • May combine two claims.

    Premisecitation

    The 5′UTR of rabbit β-globin (Rabb) consists of 52 nucleotides and does not contain strong secondary structures.

    “In contrast, the 5′UTR of rabbit β-globin (Rabb) consists of 52 nucleotides and does not contain strong secondary structures [37].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Kozak, 1994, Features in the 5′ non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency, J. Mol. Biol. source

  • May combine two claims.

    Interpretationown data

    The luciferase overexpression observed with the UTR combinations was mediated primarily by enhanced translational efficiency and to a lesser extent by increased stability of the RNA molecules.

    “Altogether, these findings indicated that the luciferase overexpression was mediated primarily by enhanced translational efficiency and to a lesser extent by increased stability of the RNA molecules.”
    • Also stated in: § 4. Discussion · plain
      “Subsequent evaluation of the number of mRNA copies at the injection site revealed that the luciferase overexpression is mediated primarily by improved translational efficiency and to a lesser extent by enhanced stability of the RNA molecules.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The number of mRNA copies at the injection site reflects the stability of the mRNA.
37 of 37

  • The paper relies on this without support.

    An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein's expression.

    “An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein’s expression.”

  • The paper relies on this without support.

    Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein's expression, which is determined by various cis- and trans-regulatory elements of the mRNA.

    “Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein’s expression, which is determined by various cis- and trans-regulatory elements of the mRNA.”

  • The paper relies on this without support.

    Cap-independent translation initiation is activated under stress.

    “On the other hand, cap-independent translation initiation is activated under stress.”

  • The paper relies on this without support.

    The number of mRNA copies at the injection site reflects the stability of the mRNA.

    “The absence of significant differences in the number of RNA copies suggested that the upregulation of luciferase was apparently not related to RNA stability.”

  • The paper relies on this without support.

    For cap-independent translation initiation involving the EMCV IRES, the binding of the J-K domain to eIF4G is needed for the recruitment of preinitiation complex 43S.

    “For cap-independent translation initiation involving the EMCV IRES, the binding of the J-K domain to eIF4G is thought to be needed; this event is necessary for the recruitment of preinitiation complex 43S.”

  • The authors state this as an assumption.

    Translation enhancement via ribosomes recruited downstream of a gene may proceed without transfer of a ribosome to the 5′ end, because the 43S recruited to the 3′UTR can find the start codon in a 5′-end-independent manner via circularization of an intermediate RNA region separating the 5′UTR and 3′UTR.

    “The authors of ref. [39] suppose that translation enhancement via the ribosomes recruited downstream of a gene may proceed (at least partially) without a transfer of a ribosome to the 5′ end, probably owing to direct recognition of a start codon by ribosomal complex 43S.”
    “In other words, the 43S recruited to the 3′UTR can find the start codon in a 5′-end-independent manner because of circularization of an intermediate RNA region separating the 5′UTR and 3′UTR.”

  • The paper relies on this without support.

    Predicted mean ribosomal loading (MRL) values are a valid indicator of translation efficiency.

    “In TPL, the presence of unique cis- and/or trans-regulatory elements that ensure efficient translation is indirectly confirmed by evidence obtained via assessment of predicted mean ribosomal loading (MRL) with the Optimus 5-Prime algorithm [46].”
7 of 7

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?

  • The interactions between 5′ and 3′ UTR are crucial and can modulate the mRNA translation efficiency.

    “The interactions between 5′ and 3′ UTR are crucial and can modulate the mRNA translation efficiency.44”

    From Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo (2025) · cites this paper as [44]

    The Discussion reports that combinations of 5′ and 3′ UTRs affected protein expression, primarily through translational efficiency. But the Conclusions describe their influence on each other as indirect, and the Introduction says they do not interact directly during translation. The claim overstates the evidence by calling their interactions crucial without that qualification. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Inclusion of specific untranslated regions is a promising strategy to enhance stability and efficacy of mRNA therapeutics.

    “Recent studies demonstrate that, in addition to the incorporation of modified nucleosides, the inclusion of specific untranslated regions represents a highly promising strategy to enhance the stability and efficacy of mRNA therapeutics [12][13][14][15][16].”

    From Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity (2026) · cites this paper as [12]

    The Introduction says UTR sequences play a key role in mRNA stability, and the Conclusions propose selected UTR combinations for therapeutic use because they increase protein expression. But the Results report no significant difference in mRNA copy numbers between groups; improved stability was only hypothesized, not demonstrated for those combinations. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • 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].”

    From Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis (2024) · cites this paper as [7]

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • ConsistentF(30, 124) = 4.105reported p < .001 · recomputed p < .001

    “F(30, 124) = 4.105, p < .001”

  • ConsistentF(30, 124) = 6.51reported p < .001 · recomputed p < .001

    “F(30, 124) = 6.51, p < .001”

  • ConsistentF(30, 124) = 5.7reported p < .001 · recomputed p < .001

    “F(30, 124) = 5.70, p < .001”

  • ConsistentF(30, 124) = 4.951reported p < .001 · recomputed p < .001

    “F(30, 124) = 4.951, p < .001”

4 of 4

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.

  • The 72-hour Kruskal-Wallis degrees of freedom do not match eight groups

    Minor

    Eight groups give a Kruskal-Wallis test 7 group degrees of freedom, not the reported 8.

    Reported 8 · recomputed 7

  • The comparison with cell data misstates the 72-hour result

    Minor

    The paper says there were no significant between-group differences at 72 hours in the cells, but its cell results report higher bioluminescence for two UTR combinations at that time point.

  • The 24-hour test reports more observations than mice

    Minor

    The in vivo experiment used 64 mice, but the reported 24-hour Kruskal-Wallis notation gives 71 as its second value. Whether that value denotes observations or total degrees of freedom, it exceeds what 64 mice could provide.

  • The reported 8-hour in vivo effect does not meet the paper's significance threshold

    Minor

    The paper says the Kruskal-Wallis test found an effect at 8 hours, but its reported p = 0.064 is above the stated significance threshold of p < 0.05.

4 of 4

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Related

Authors
Kirshina, A., Vasileva, O., Kunyk, D., Seregina, K., Muslimov, A., Ivanov, R., & Reshetnikov, V.
Affiliations
Sirius University of Science and Technology (RU)
Corresponding author
Reshetnikov, Vasiliy
ORCID
Venue
Biomolecules
Publisher
MDPI AG
Published
2023-11-20
Volume
13
Issue
11
Pages
1677
ISSN
2218-273X (ISSN-L)
DOI
10.3390/biom13111677
PMC
PMC10669451
OpenAlex
W4388823645
Semantic Scholar
734cfbcb19f9139d8ec2ee7c34d697200d910e20
Cited by
26
References
52
Citations by year
2026: 8 · 2025: 8 · 2024: 9 · 2023: 1
FWCI
2.25
Type
journal article
Language
en
Open access
Licence
Locations
  • Biomolecules (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
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 Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
5' untranslated region, 3' untranslated region, mRNA translation efficiency, mRNA stability, firefly luciferase, in vitro transcribed RNA, DC2.4 cells, mRNA vaccines
MeSH terms
Animals, Luciferases, Rabbits, RNA, Messenger, Transfection, Protein Biosynthesis, 5' Untranslated Regions, 3' Untranslated Regions, Mice

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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