A Short 63-Nucleotide Element Promotes Efficient circRNA Translation

  1. Martina Chiara Biagi
  2. Andrea Giuliani
  3. Alessia Grandioso
  4. Irene Bozzoni
  5. Gaia Di Timoteo

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Abstract

Circular RNAs (circRNAs) are a class of RNA with multiple functions, including the ability to be translated.

Several intrinsic features of circRNAs, such as high stability, confer them advantages over linear RNAs; therefore, circRNA-based drugs have recently received increasing attention.

However, the inefficiency of their cap-independent translation and the difficulties in the large-scale production of long circRNAs negatively impact on their use in therapy. Some efforts have been done to solve these issues related to circRNA adoption, but, to date, circRNA translation still relies on long IRESs (600-800) and chemical group addition. In this study, identified a 63-nt element able to drive circRNA translation comparably to the most commonly used IRESs. This element includes a a 13-nt sequence previously reported to enhance linear RNA translation and a segment of the UTR of the endogenously translated circRNA circZNF609. Notably, this element combines a comparable IRES-like efficiency to a considerably shorter length, expanding the landscape of ORFs potentially suitable for being translated from circRNAs and enhancing their potential as therapeutic agents in therapy.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    REVIEWER 1

    This is an important and solid study that identified sequences that can improve circRNA translation and that as or more importantly are very short and hence are suitable for generating of efficient protein expressing circRNAs. This manuscript fills an important gap in the field, and it is highly significant. The study is well controlled, the rationale clear and the results conclusive with no major flaws.

    • While this is a minor concern as the vector has been used before, it will greatly improve the quality of the paper if the authors could just verify that the vector only generates circRNA molecules and not linear concatenamers. To do so the authors can focus only in their control and the most optimal transcripts and perform northern blot or well controlled RNAseR experiments to show that all RNA molecules containing the back splicing junction are circular *We thank the reviewer for raising this point. As suggested, we performed RNaseR resistance assays on our three most efficient candidates driving cGFP translation (VCIP, T3-glo, and T3-U3) to confirm that all derived RNA molecules containing the back-splicing junction are circular. **As proof of this, cGFP proved strongly resistant to RNase R (new Fig. S1N), confirming its circular structure. **We further ruled out the possibility that molecules other than the circRNA encoding GFP serve as templates for translation from our vectors. Specifically, ad hoc PCR amplifications performed for this purpose (new Fig. S1M) showed no bands that would indicate the presence of concatemers. Indeed, ad hoc PCR amplifications (new Fig. S1M) revealed no bands indicative of concatemer formation. The primers used and the expected sizes of the amplicons are schematically represented in new Fig. S1M. In brief, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF, thus detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed (new Fig. S1M). Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively. **These results are expected for a circRNA, as also indicated by the fact that the circZNF609 positive control behaves in a similar manner. *Collectively, these results confirm the circular nature of our transcript and exclude translation originating from possible concatemers.
    • **These results are shown in new Fig. S1M and S1N and described in the text as follows: “Importantly, we ruled out the possibility that templates other than the GFP-encoding circRNA drive translation from our best performing constructs (V-cGFP, T3-glo-cGFP and T3-U3-cGFP). Ad hoc PCRs amplifications (Fig. S1M) revealed no bands indicative of concatemer formation. The left panel of Fig. S1M schematically illustrates the primer sets and expected amplicons sizes. In particular, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed. *Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively (Fig. S1M). These results are consistent with the circularity of the transcripts tested and coherent with the results obtained for circZNF609, used as control (Fig. S1M). Finally, cGFP resulted resistant to RNAseR treatment (Fig. S1N), further supporting its circular nature.”

    • There is a repetition of the world "a" in the abstract. We thank the reviewer for the attention paid to our text, we removed the extra “a” from the abstract.

    • All circRNA translation studies should be cited when describing translation of circRNAs. We thank the reviewer for the suggestions, we corrected the mistake present in the text and included extra referenced about circRNA translation.

    *Specifically, we included: *

    • *Fan, X., Yang, Y., Chen, C. et al. Pervasive translation of circular RNAs driven by short IRES-like elements. Nat Commun 13, 3751 (2022). *https://doi.org/10.1038/s41467-022-31327-y
    • Chen CK et al. Structured elements drive extensive circular RNA translation. Mol Cell. 2021 Oct 21; 81(20):4300-4318.e13.doi: 10.1016/j.molcel.2021.07.042. Epub 2021 Aug 25. PMID: 34437836; PMCID: PMC8567535.
    • Obi P, Chen YG. The design and synthesis of circular RNAs. Methods. 2021 Dec;196:85-103. doi: 10.1016/j.ymeth.2021.02.020. Epub 2021 Mar 2. PMID: 33662562; PMCID: PMC8670866.
    • *Fukuchi, K., Nakashima, Y., Abe, N. et al. Internal cap-initiated translation for efficient protein production from circular mRNA. Nat Biotechnol (2025). *https://doi.org/10.1038/s41587-025-02561-8
    • *Du, Y., Zuber, P.K., Xiao, H. et al. Efficient circular RNA synthesis for potent rolling circle translation. Nat. Biomed. Eng 9, 1062–1074 (2025). *https://doi.org/10.1038/s41551-024-01306-3
    • Wang F, Cai G, Wang Y, Zhuang Q, Cai Z, Li Y, Gao S, Li F, Zhang C, Zhao B, Liu X. Circular RNA-based neoantigen vaccine for hepatocellular carcinoma immunotherapy. MedComm (2020). 2024 Jul 29;5(8):e667. doi: 10.1002/mco2.667. PMID: 39081513; PMCID: PMC11286538.
    • Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3. PMID: 26342664.
    • Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen LL, Wang Y, Wong CC, Xiao X, Wang Z. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res. 2017 May;27(5):626-641. doi: 10.1038/cr.2017.31. Epub 2017 Mar 10. PMID: 28281539; PMCID: PMC5520850. REVIEWER 2

    Circular RNAs (circRNAs) have attracted significant interest due to their unique properties, which make them promising tools for expressing exogenous proteins of therapeutic value. However, several limitations must be addressed before circRNAscan become a biologically and economically viable platform for the biotech industry.One of the main challenges is the reliance on large, highly structured sequences withinternal ribosome entry site (IRES) activity to initiate translation of the downstream open reading frame. In this study, the authors propose an alternative strategy that combines the 5′ untranslated region (5′UTR) of a previously characterized natural circRNA(circZNF609) with a short 13-nt nucleotide sequence shown to act as a translational enhancer. By evaluating the activity of various constructs containing a reporter geneacross multiple cell lines, they identify the most efficient and compact sequence, 63-nt long, capable of boosting translation within a circular RNA context.

    Major Comments:

    • This study is well-executed and relies on standard in vitro molecular biology techniques, which are adequate to support the conclusions drawn. *We thank the reviewer for the very positive opinion on the execution of our study. *

    • The experimental procedures are clearly described, and the statistical analyses have been performed according to accepted standards. *We thank the reviewer for the very positive comment about the analyses we performed. *

    Minor Comments:

    • The manuscript would greatly benefit from a comprehensive revision to improve clarity and language. Involving a native English speaker during the editing process could significantly enhance the manuscript's readability and overall quality. The Results section would benefi t from closer attention, as certain parts of the description are attimes confusing and could be clarifi ed for better reader comprehension. We thank the reviewer for the input. We performed a huge revision of the text to improve language quality and enhance readability. We extended the descriptions in the results sections in order to explicit and clarify our data.

    • The references should be carefully reviewed for accuracy and consistency-forinstance, references 9 and 10 appear to require correction or clarifi cation. We thank the reviewer for the careful reading of our paper. We amended the reference section, and we expanded it.

    Reviewer #2 (Significance (Required)):

    This study addresses a critical bottleneck in RNA therapeutics. The use of the proposed short sequences could significantly enhance the in vivo activity of protein-encoding circular RNAs. A highly efficient, compact translational enhancer has thepotential to substantially improve the therapeutic applicability of circRNAs and broaden their range of applications. Given the potential utility of these findings, we would anticipate pursuing intellectual property (IP) protection. To further strengthen the study, future work should include additional data on polysome association and a detailed analysis of the secondary structure of the 66-nt enhancer sequence. This work should be of broad interest to molecular biologists working on RNA biology, translation, and RNA-based therapeutics. I expect the identified sequence will betested by multiple laboratories to evaluate its strength and versatility, further underscoring the potential impact of this study. For context, I am actively engaged in research on non-coding RNAs.

    REVIEWER 3

    In this brief report, the authors take advantage of circular RNA expression plasmids to define elements that can be used to enable efficient translation. They test a handful of known IRES elements as well as short translation enhancing elements (TEEs) for their ability to promote translation of circular GFP and c-ZNF609 reporters. They focus on one particular element that is of a short length and seems to work as well as longer IRES elements. My major concern relates to possible alternative sources of the translated proteins, which the authors have not ruled out (see below). I find themanuscript to be too preliminary in its current state.

    • Work from the Meister group (Ho-Xuan et al 2020 Nucleic Acids Res 48:10368) has shown that apparent translation from circRNA over-expression plasmids is not from circular RNAs, but instead from trans-splicing linear by-products. The authors have not ruled out such alternative explanations here, e.g. by using deletion constructs that prevent backsplicing. *We thank the reviewer for raising this point. **We ruled out the possibility that molecules other than the circRNA encoding GFP serve as templates for translation from our vectors. Specifically, ad hoc PCR amplifications performed for this purpose (new Fig. S1M) showed no bands that would indicate the presence of concatemers. Indeed, ad hoc PCR amplifications (new Fig. S1M) revealed no bands indicative of concatemer formation. The primers used and the expected sizes of the amplicons are schematically represented in new Fig. S1M. In particular, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed. Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively. These results are expected for a circRNA, as also indicated by the fact that the circZNF609 positive control behaves in a similar manner. Collectively, these results confirmed the circular nature of our transcript and excluded translation originating from possible concatemers. *

    *These results are shown in new Fig. S1M and S1N and described in the text as follows: “Importantly, we ruled out the possibility that templates other than the GFP-encoding circRNA drive translation from our top constructs (V-cGFP, T3-glo-cGFP and T3-U3-cGFP). Ad hoc PCRs amplifications (Fig. S1M) revealed no bands indicative of concatemer formation. The left panel of Fig. S1M schematically illustrates the primer sets and expected amplicons sizes. In brief, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed (new Fig. S1M). **Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively (Fig. S1M). These results are consistent with the circularity of the transcripts tested (Fig. S1M). **Importantly, cGFP PCR amplifications showed similar results as a validated endogenous circRNA, namely circZNF609, used as control (Fig. S1M, right panel), confirming the circular nature of cGFP. Finally, cGFP resulted resistant to RNAseR treatment (Fig. S1N), further supporting its circular nature.” *

    • Echoing the point above, the overall results would be stronger if the authors couldconfirm IRES activity using highly pure, in vitro transcribed RNAs that are transfected into cells* * We thank the reviewer for this suggestion. Unfortunately, we are currently unable to produce synthetic circular molecules in-house, and the cost and time for purchasing synthetic ones are prohibitive. Nevertheless, we have performed the experiments described above to ensure the circularity of the transcripts tested.

    • The authors should also confirm their IRES activity using standard dual luciferase reporter (linear) constructs which have long been a standard approach in the field. *We thank the reviewer for raising this point. As recommended, we cloned our three best candidates (VCIP, T3-glo, and T3-U3) into the pRL-TK/pGL3 dual-luciferase vector to assess their IRES activity (producing the vectors VCIP-Luc, T3-glo-Luc, and T3-U3-Luc), transfected them into RD cells, and, after 24 h of incubation, measured luciferase activity to assess the IRES performance of each candidate. From our analyses, VCIP and T3-U3 confirmed their IRES activity, although showing different relative efficiency, whereas T3-glo was inactive in the linear luciferase context. This finding is consistent with previous observations (Legnini et al., 2017) showing that the performance of IRES sequences in a linear luciferase reporter may differ from their activity when driving translation from a circRNA template. Overall, these results highlight the need for further investigation into the sequences *and contexts *specifically governing circRNA translation, rather than relying solely on knowledge derived from linear RNAs. **The results are shown below. We did not include them in the text to not overcomplicate the readability. However, we are happy to add and discuss them if required. *

    ***

    ***

    *Bar plot representing the relative luciferase activity deriving from VCIP-Luc (“V”), T3-glo-Luc (“T3-glo”), and T3-U3-Luc (“T3-U3”). Dual luciferase assay was performed and Renilla luciferase activity from each candidate was normalized against the Firefly luciferase. An empty ptKRL-pgl3 vector was used as reference. The ratio of each sample versus its experimental control was tested by two-tailed Student’s t test. * indicates a Student’s t test-derived p-value * *

    • Methods, Plasmids Construction Section: Rather than including long lists of oligos and forcing a reader to figure out the final product that was cloned, it would be more intuitive if the authors provided the full sequences of the ORF and IRES sequencesthat were tested. We thank the reviewer for the comment, we added the sequences to the methods (Supplementary Table 1).

    • The manuscript needs extensive English editing. Parts of it are also formatted in anunusual style, especially the introduction where it seems like each paragraph is a single sentence. As requested by the reviewer, we edited the text to make the language and content more accessible to readers.

    • References included by the authors are selective and surprisingly do not include Chen et al (2021) Mol Cell 20:4300-4318 which already defined IRES elements for circRNAs that are fairly small. *Thank you for pointing this out. We have now cited the elegant work of Chen et al. (2021, Mol Cell 20:4300–4318) in the revised manuscript. While Chen and colleagues screened IRES-like elements of roughly 200 nt, our study was designed to uncover an even more minimal motif. The elements we report are therefore markedly shorter, highlighting a complementary, rather than overlapping, aspect of IRES available for driving circRNA translation. However, we now refer to Chen et al. in our text. *

    • Error bars in Fig 2, especially Fig 2B, are huge. It seems impossible to make any conclusion given the large variety across these experiments. *Thank you for your input. Although the error bars appear relatively large, the overall conclusions remain robust, as also noted by the other reviewers: both T3-glo and T3-U3 are intrinsically compact elements, yet they drive translation as efficiently as larger canonical IRESs. The error bars largely reflect the inherent variability of transient transfection assays, which naturally increases with the number of constructs examined. **To strengthen our dataset without discarding existing replicates, we chose not to repeat experiments in the previously tested lines. Instead, we assessed our vectors in an additional model, the D283 medulloblastoma cell line. In this setting, we unexpectedly observed that the EMCV IRES surpasses the VCIP IRES, opposite to what we saw in the other lines, yet even here the short elements we identified remain strong competitors (new Fig. 2C, S2G, S2H). *The evaluation of multiple CDSs across several cell lines, make our findings to be solid and well supported.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this brief report, the authors take advantage of circular RNA expression plasmids to define elements that can be used to enable efficient translation. They test a handful of known IRES elements as well as short translation enhancing elements (TEEs) for their ability to promote translation of circular GFP and c-ZNF609 reporters. They focus on one particular element that is of a short length and seems to work as well as longer IRES elements. My major concern relates to possible alternative sources of the translated proteins, which the authors have not ruled out (see below). I find the manuscript to be too preliminary in its current state.

    Major comments:

    • Work from the Meister group (Ho-Xuan et al 2020 Nucleic Acids Res 48:10368) has shown that apparent translation from circRNA over-expression plasmids is not from circular RNAs, but instead from trans-splicing linear by-products. The authors have not ruled out such alternative explanations here, e.g. by using deletion constructs that prevent backsplicing.
    • Echoing the point above, the overall results would be stronger if the authors could confirm IRES activity using highly pure, in vitro transcribed RNAs that are transfected into cells.
    • The authors should also confirm their IRES activity using standard dual luciferase reporter (linear) constructs which have long been a standard approach in the field.
    • Methods, Plasmids Construction Section: Rather than including long lists of oligos and forcing a reader to figure out the final product that was cloned, it would be more intuitive if the authors provided the full sequences of the ORF and IRES sequences that were tested.
    • The manuscript needs extensive English editing. Parts of it are also formatted in an unusual style, especially the introduction where it seems like each paragraph is a single sentence.
    • References included by the authors are selective and surprisingly do not include Chen et al (2021) Mol Cell 20:4300-4318 which already defined IRES elements for circRNAs that are fairly small.
    • Error bars in Fig 2, especially Fig 2B, are huge. It seems impossible to make any conclusion given the large variety across these experiments.

    Minor comments:

    • Provide a reference for the claim in the introduction that "the smaller the RNA to be circularized, the greater the circularization efficiency".
    • Supplemental Table: Please clarify what each qPCR primer was used for. E.g. what does "49 hung rev" refer to?
    • Fig 1C should be explained better. What do the numbers in white refer to? In the main text, it is written that "Furthermore, we also added TEEs elements upstream the VCIP IRES" but Fig 1C suggests they were inserted downstream.

    Referees cross-commenting

    I stand by my comments regarding the need for the authors to perform additional controls and validation.

    Significance

    This work is most relevant for researchers aiming to use circular RNAs as therapeutic modalities to express proteins. Defining optimal methods, including IRES elements, that enable maximal translational output would be helpful. Note, however, that this is far from the first study to look for IRES elements in circular RNAs (e.g. Chen et al (2021) Mol Cell 20:4300-4318) which did it in a much more extensive manner.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Circular RNAs (circRNAs) have attracted significant interest due to their unique properties, which make them promising tools for expressing exogenous proteins of therapeutic value. However, several limitations must be addressed before circRNAs can become a biologically and economically viable platform for the biotech industry. One of the main challenges is the reliance on large, highly structured sequences with internal ribosome entry site (IRES) activity to initiate translation of the downstream open reading frame. In this study, the authors propose an alternative strategy that combines the 5′ untranslated region (5′UTR) of a previously characterized natural circRNA (circZNF609) with a short 13-nt nucleotide sequence shown to act as a translational enhancer. By evaluating the activity of various constructs containing a reporter gene across multiple cell lines, they identify the most efficient and compact sequence, 63-nt long, capable of boosting translation within a circular RNA context.

    Major Comments:

    • This study is well-executed and relies on standard in vitro molecular biology techniques, which are adequate to support the conclusions drawn.
    • The experimental procedures are clearly described, and the statistical analyses have been performed according to accepted standards.

    Minor Comments:

    • The manuscript would greatly benefit from a comprehensive revision to improve clarity and language. Involving a native English speaker during the editing process could significantly enhance the manuscript's readability and overall quality. The Results section would benefit from closer attention, as certain parts of the description are at times confusing and could be clarified for better reader comprehension.
    • The references should be carefully reviewed for accuracy and consistency-for instance, references 9 and 10 appear to require correction or clarification.

    Significance

    This study addresses a critical bottleneck in RNA therapeutics. The use of the proposed short sequences could significantly enhance the in vivo activity of protein-encoding circular RNAs. A highly efficient, compact translational enhancer has the potential to substantially improve the therapeutic applicability of circRNAs and broaden their range of applications.

    Given the potential utility of these findings, we would anticipate pursuing intellectual property (IP) protection.

    To further strengthen the study, future work should include additional data on polysome association and a detailed analysis of the secondary structure of the 66-nt enhancer sequence.

    This work should be of broad interest to molecular biologists working on RNA biology, translation, and RNA-based therapeutics. I expect the identified sequence will be tested by multiple laboratories to evaluate its strength and versatility, further underscoring the potential impact of this study.

    For context, I am actively engaged in research on non-coding RNAs.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript entitled "A Short 63-Nucleotide Element Promotes Efficient circRNA Translation", Biagi et al. aim to identify sequences and layouts that would allow high expression of proteins from an engineered circular RNA (circRNA). Briefly, the authors utilize a circRNA-producing plasmid that produces a GFP protein encoded across the splice junction when translated and test different IRESs in combination with Translation Enhancing Element (TEEs). While performing these experiments they found that a short sequence containing the TEE (13-glo) is enough to promote significant levels of translation while keeping the size of the circRNA small. The authors then tested whether the presence of a spacer could help improving translation and identified a 50base sequence that in combination with the TEE can promote very efficient translation. The authors then went on and showed that this element can promote the translation from a circRNA expressing another protein (in this case was a circRNA-encoded peptide), demonstrating the versatility of this approach. Moreover, the authors showed that their approach can promote translation in other cell lines.

    This is an important and solid study that identified sequences that can improve circRNA translation and that as or more importantly are very short and hence are suitable for generating of efficient protein expressing circRNAs. This manuscript fills an important gap in the field, and it is highly significant. The study is well controlled, the rationale clear and the results conclusive with no major flaws. While this is a minor concern as the vector has been used before, it will greatly improve the quality of the paper if the authors could just verify that the vector only generates circRNA molecules and not linear concatamers. To do so the authors can focus only in their control and the most optimal transcripts and perform northern blot or well controlled RNAseR experiments to show that all RNA molecules containing the back splicing junction are circular.

    Minor comments:

    • There is a repetition of the world "a" in the abstract.
    • All circRNA translation studies should be cited when describing translation of circRNAs.

    Significance

    While other studies have identified sequences that can drive circRNA translation, this study has done a great job identifying a very short sequence and additional requirements for optimal translation. This is an important study that will be of high interest for the molecular, cell biology and general biology communities.

  5. Version published to 10.1101/2025.05.13.653105 on bioRxiv