ISSN 0253-2778

CN 34-1054/N

Open AccessOpen Access JUSTC Life Sciences 28 September 2023

Modulating miRNA binding sites within circRNA for enhanced translation efficiency

Cite this:
https://doi.org/10.52396/JUSTC-2023-0048
More Information
  • Author Bio:

    Kewei Zhang is currently a master’s student at the Division of Life Sciences and Medicine, University of Science and Technology of China, under the supervision of Prof. Ge Shan. Her research mainly focuses on circular RNAs and the applications

    Ge Shan is currently a Professor at the Division of Life Sciences and Medicine, University of Science and Technology of China. His research interests include functions and functional mechanisms of noncoding RNAs

    Liang Chen is currently a Research Professor at the Division of Life Sciences and Medicine, University of Science and Technology of China. His research interests include noncoding RNAs and their links to human diseases

  • Corresponding author: E-mail: shange@ustc.edu.cn; E-mail: anqingcl@ustc.edu.cn
  • Received Date: 19 March 2023
  • Accepted Date: 25 June 2023
  • Available Online: 28 September 2023
  • Circular RNAs (circRNAs) are covalently closed circular RNAs, and some of them preserve translation potency. However, modulation of circRNA translation efficiency and its applications need to be explored. In this study, RNAs containing the translation initiation element CVB3 IRES and the coding sequence of luciferase protein were transcribed and circularized in vitro by T7 RNA polymerase and an optimized permutated intron‒exon (PIE) splicing strategy. The circularized RNAs were then transfected and translated into active luciferase in the cultured cells. Insertion of miRNA binding sites at the flanking region of the luciferase coding sequence significantly reduced the translation efficiency of the circRNAs. Mutations of the miRNA binding sites in the firefly luciferase coding sequence led to increased translation efficiency of synthetic circRNAs in cells. We also proved that mutations of the binding sites of specific miRNAs also enhanced the translation efficiency of synthetic circRNAs. Further in vivo experiments via bioluminescence imaging showed that synonymous mutation of the miRNA binding sites promoted synthetic circRNA translation in nude mice. This study demonstrates that the modulation of miRNA binding sites affects the translation efficiency of synthetic circRNAs in vitro and in vivo, which could be used as versatile tools for future applications in clinical imaging.
    Modulating miRNA binding sites within circRNAs can effectively enhance their translation efficiency both in vitro and in vivo.
    Circular RNAs (circRNAs) are covalently closed circular RNAs, and some of them preserve translation potency. However, modulation of circRNA translation efficiency and its applications need to be explored. In this study, RNAs containing the translation initiation element CVB3 IRES and the coding sequence of luciferase protein were transcribed and circularized in vitro by T7 RNA polymerase and an optimized permutated intron‒exon (PIE) splicing strategy. The circularized RNAs were then transfected and translated into active luciferase in the cultured cells. Insertion of miRNA binding sites at the flanking region of the luciferase coding sequence significantly reduced the translation efficiency of the circRNAs. Mutations of the miRNA binding sites in the firefly luciferase coding sequence led to increased translation efficiency of synthetic circRNAs in cells. We also proved that mutations of the binding sites of specific miRNAs also enhanced the translation efficiency of synthetic circRNAs. Further in vivo experiments via bioluminescence imaging showed that synonymous mutation of the miRNA binding sites promoted synthetic circRNA translation in nude mice. This study demonstrates that the modulation of miRNA binding sites affects the translation efficiency of synthetic circRNAs in vitro and in vivo, which could be used as versatile tools for future applications in clinical imaging.
    • A circular RNA capable of expressing firefly luciferase and renilla luciferase was constructed.
    • Binding sites for miRNAs within the circular RNA decrease its translation efficiency without affecting its stability.
    • Synonymous mutations in the miRNA binding sites can enhance the translation efficiency of the circular RNA without affecting its stability.
    • In vivo experiments using nude mice and cellular-level experiments both demonstrate that reducing specific miRNA binding sites can improve the translation efficiency and expression duration of the circular RNA.

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  • JUSTC-2023-0048 Supporting information.zip
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Catalog

    Figure  1.  Protein expression initiated by circRNAs. (a, b) Schematic representation of the elements required for circFLuc (a) and circRLuc (b) production through the PIE system. (c) Agarose gel showing linear RNA after in vitro transcription (IVT) and untreated and treated in vitro cyclization (IVC) RNA products with RNase R. (d) Sanger sequencing chromatogram for the junction site of the reverse-transcribed circFLuc and circRLuc samples. (e) Luciferase activity (RLU) of circFLuc and circRLuc in HEK293T cells. Statistical analysis of luciferase activity was performed at 24 h posttransfection. FLuc represents firefly luciferase. RLuc represents Renilla luciferase. RLU, relative light unit.

    Figure  2.  Translation efficacy of circFLuc(miR) and circFLuc. (a) Heatmap displaying the expression profile of miRNAs in human healthy tissues. Data were normalized using the Z-score method. (b) Relative expression levels of microRNAs in the database were determined by RT‒qPCR, with U6 RNA serving as an internal control (n = 3). (c) Schematic diagram of the circularized sequences after the addition of miRNA binding sites (miR-21-5p, miR-101-3p, miR-192-5p). (d) Agarose gel showing linear RNA after in vitro transcription (IVT) and untreated and treated in vitro cyclization (IVC) RNA products with RNase R. (e) Luciferase activity (RLU) of circFLuc(miR), circFLuc, and circRLuc in HEK293T cells. Activity was measured at 24 h posttransfection. (f) Relative luciferase activity of 293T cells transfected with circFLuc(miR) and circFLuc, with cotransfected circRLuc serving as an internal control, and relative firefly luciferase activity normalized to RLuc (n = 4). (g) Relative circRNA levels in HEK293T cells transfected with circFLuc(miR) and circFLuc, normalized to GAPDH mRNA and circRLuc (n = 4). Statistical analyses were performed at different time points. RLU, relative light unit. ns represents no significant difference, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Figure  3.  Translation efficacy of circFLuc(mut) and circFLuc. (a) Schematic illustration of circularized sequences with synonymous mutations introduced in miRNA binding sites. (b) Agarose gel showing linear RNA after in vitro transcription (IVT) and untreated and treated in vitro cyclization (IVC) RNA products with RNase R. (c) Luciferase activity (RLU) of circFLuc(mut), circFLuc and circRLuc in HEK293T cells at 24 h posttransfection. (d–g) Left panel shows the relative luciferase activity of HEK293T cells transfected with circFLuc(mut) or circFLuc (n = 3). CircRLuc was used as an internal control for cotransfection, while miR-129-5p and miR-362-5p were overexpressed through mimics (mimic). The nonoverexpressing group was transfected with NC-mimics as a control (NC). The relative luciferase activity was standardized using RLuc activity. The right panel shows the relative expression level of circRNAs in transfected cells (n = 3) normalized to actin mRNA and circRLuc. Statistical analysis was performed at 24 h posttransfection. RLU, relative light unit. ns, not significant, *p < 0.05, ** p < 0.01.

    Figure  4.  Translation efficacy of circFLuc(mut miR) and circFLuc. (a) Schematic diagram of the circularized sequence with synonymous mutations in the miR-129-5p and miR-362-5p binding sites. (b) Agarose gel showing linear RNA after in vitro transcription (IVT) and untreated and treated in vitro cyclization (IVC) RNA products with RNase R. (c) Luciferase activity (RLU) of circFLuc(mut miR), circFLuc, and circRLuc in HEK293T cells, with detection of luciferase activity at 24 h posttransfection. (d–g) The left panel shows the relative luciferase activity of HEK293T cells (n = 3) transfected with circFLuc(mut miR) or circFLuc. CircRLuc was used as an internal control for cotransfection, while miR-129-5p and miR-362-5p were overexpressed through plasmids (OE). The nonoverexpressing group was transfected with an empty plasmid backbone as a control (EV). The relative luciferase activity was standardized using RLuc activity. The right panel shows that the relative circRNA level was standardized using actin mRNA and circRLuc. Statistical analysis was performed at 24 h posttransfection. RLU, relative light unit. ns, no significance, *p < 0.05, ** p < 0.01.

    Figure  5.  In vivo translation efficiency of circFLuc(mut miR) and circFLuc. (a) Schematic diagram of the in vivo experiment in nude mice. (b) Bioluminescence imaging of HepG2 cells (top), HepG2-circFLuc cells (middle), and HepG2-circFLuc(mut miR) cells (bottom) was performed. Luciferase activity was detected 24 h after transfection of different cell numbers. Images show color-coded maps of photon flux superimposed on black-and-white photographs of the assay plates. (c) The statistical results of (b) show that the bioluminescence values of circFLuc(mut miR) and circFLuc are linearly correlated with the number of cells, with r2 values of 0.9965 and 0.9954, respectively. Luminescence values in the mock group did not change with increasing cell number. (d) In vivo bioluminescence image of mice subcutaneously injected with HepG2-circFLuc (top) and HepG2-circFLuc(mut miR) (bottom) and measured for luciferase activity 2 d post-injection (n = 6 per group, 106 cells per mouse). (e) Statistical analysis of the in vivo luminescence values from (d). (f) In vivo bioluminescence image of mice subcutaneously injected with HepG2-circFLuc (top) and HepG2-circFLuc(mut miR) (bottom) and measured for luciferase expression 4 d post-injection (n = 6 per group, 106 cells per mouse). (g) Statistical analysis of the in vivo luminescence values from (f). ns, no significance, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Figure  6.  A simplified summary of this study, in vitro synthesis of circRNA and related changes in miRNA binding sites and their roles in vitro and in vivo.

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    [2]
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    [3]
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    [4]
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    [7]
    Barrett S P, Salzman J. Circular RNAs: analysis, expression and potential functions. Development, 2016, 143 (11): 1838–1847. doi: 10.1242/dev.128074
    [8]
    Legnini I, Di Timoteo G, Rossi F, et al. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Molecular Cell, 2017, 66 (1): 22–37.e9. doi: 10.1016/j.molcel.2017.02.017
    [9]
    Pamudurti N R, Bartok O, Jens M, et al. Translation of CircRNAs. Molecular Cell, 2017, 66 (1): 9–21.e7. doi: 10.1016/j.molcel.2017.02.021
    [10]
    Li H, Peng K, Yang K, et al. Circular RNA cancer vaccines drive immunity in hard-to-treat malignancies. Theranostics, 2022, 12 (14): 6422–6436. doi: 10.7150/thno.77350
    [11]
    Qu L, Yi Z, Shen Y, et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell, 2022, 185 (10): 1728–1744.e16. doi: 10.1016/j.cell.2022.03.044
    [12]
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    [13]
    Chen C K, Cheng R, Demeter J, et al. Structured elements drive extensive circular RNA translation. Molecular Cell, 2021, 81 (20): 4300–4318.e13. doi: 10.1016/j.molcel.2021.07.042
    [14]
    Fan X, Yang Y, Chen C, et al. Pervasive translation of circular RNAs driven by short IRES-like elements. Nature Communications, 2022, 13 (1): 3751. doi: 10.1038/s41467-022-31327-y
    [15]
    Chen R, Wang S K, Belk J A, et al. Engineering circular RNA for enhanced protein production. Nature Biotechnology, 2023, 41 (2): 262–272. doi: 10.1038/s41587-022-01393-0
    [16]
    Memczak S, Jens M, Elefsinioti A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature, 2013, 495 (7441): 333–338. doi: 10.1038/nature11928
    [17]
    Piwecka M, Glažar P, Hernandez-Miranda L R, et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science, 2017, 357 (6357): eaam8526. doi: 10.1126/science.aam8526
    [18]
    Weng W, Wei Q, Toden S, et al. Circular RNA ciRS-7—A promising prognostic biomarker and a potential therapeutic target in colorectal cancer. Clinical Cancer Research, 2017, 23 (14): 3918–3928. doi: 10.1158/1078-0432.CCR-16-2541
    [19]
    Kleaveland B, Shi C Y, Stefano J, et al. A network of noncoding regulatory RNAs acts in the mammalian brain. Cell, 2018, 174 (2): 350–362.e17. doi: 10.1016/j.cell.2018.05.022
    [20]
    Hansen T B, Wiklund E D, Bramsen J B, et al. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. The EMBO Journal, 2011, 30 (21): 4414–4422. doi: 10.1038/emboj.2011.359
    [21]
    Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nature Reviews Genetics, 2010, 11 (9): 597–610. doi: 10.1038/nrg2843
    [22]
    Treiber T, Treiber N, Meister G. Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nature Reviews Molecular Cell Biology, 2019, 20 (1): 5–20. doi: 10.1038/s41580-018-0059-1
    [23]
    Bartel D P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 2004, 116 (2): 281–297. doi: 10.1016/S0092-8674(04)00045-5
    [24]
    Bartel D P. Metazoan microRNAs. Cell, 2018, 173 (1): 20–51. doi: 10.1016/j.cell.2018.03.006
    [25]
    Griffiths-Jones S, Grocock R J, van Dongen S, et al. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research, 2006, 34 (suppl_1): D140–D144. doi: 10.1093/nar/gkj112
    [26]
    Zhou W Y, Cai Z R, Liu J, et al. Circular RNA: metabolism, functions and interactions with proteins. Molecular Cancer, 2020, 19 (1): 172. doi: 10.1186/s12943-020-01286-3
    [27]
    Puttaraju M, Been M D. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Research, 1992, 20 (20): 5357–5364. doi: 10.1093/nar/20.20.5357
    [28]
    Ford E, Ares M Jr. Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91 (8): 3117–3121. doi: 10.1073/pnas.91.8.3117
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    Sokolova N I, Ashirbekova D T, Dolinnaya N G, et al. Chemical reactions within DNA duplexes. Cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent. FEBS Letters, 1988, 232 (1): 153–155. doi: 10.1016/0014-5793(88)80406-X
    [30]
    Micura R. Cyclic oligoribonucleotides (RNA) by solid-phase synthesis. Chemistry–A European Journal, 1999, 5 (7): 2077–2082. doi: 10.1002/(SICI)1521-3765(19990702)5:7%3C2077::AID-CHEM2077%3E3.0.CO;2-U
    [31]
    Miller E S, Kutter E, Mosig G, et al. Bacteriophage T4 genome. Microbiology and Molecular Biology Reviews, 2003, 67 (1): 86–156. doi: 10.1128/MMBR.67.1.86-156.2003
    [32]
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