https://doi.org/10.4081/jbr.2026.14236
An exogenous reporter system reveals location-specific miRNA regulatory modes: translational blockade at CDS vs transcript degradation at 3'UTR
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Published: 8 June 2026
MicroRNA (miRNA) is a key regulator of gene expression, involved in modulating various physiological and pathological processes. Current research primarily focuses on the regulatory mechanisms of miRNA targeting the 3' untranslated region (3'UTR), while understanding of its functional mode when targeting the protein-coding sequence (CDS) remains limited. This study established an exogenous gene expression system to systematically compare the regulatory differences in gene expression between the miRNA-3'UTR and miRNA-CDS targeting modes. Western blotting and fluorescent reporter analyses confirmed that both modes effectively suppressed protein expression, but miRNA-mediated suppression targeting CDS responded more rapidly. Real-Time Polymerase Chain Reaction (RT-PCR) analysis further revealed that the miRNA-3'UTR mode led to significant downregulation of mRNA levels, whereas the miRNA-CDS mode induced only a transient and mild reduction in mRNA. Collectively, these results demonstrate that the mechanism of miRNA action is position-dependent: targeting the 3'UTR primarily induces mRNA degradation, while targeting the CDS preferentially inhibits the translation process. This model provides an experimental framework for deciphering new mechanisms of miRNA-mediated gene expression regulation and lays a foundation for functional studies of non-coding RNAs and related disease mechanisms.
Downloads
1. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522-31. DOI: https://doi.org/10.1038/nrg1379
2. Chipman LB, Pasquinelli AE. miRNA Targeting: Growing beyond the seed. Trends Genet 2019;35:215-22. DOI: https://doi.org/10.1016/j.tig.2018.12.005
3. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281-97. DOI: https://doi.org/10.1016/S0092-8674(04)00045-5
4. Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol 2007;23:175-205. DOI: https://doi.org/10.1146/annurev.cellbio.23.090506.123406
5. Flynt AS, Lai EC. Biological principles of microRNA-mediated regulation: shared themes amid diversity. Nat Rev Genet 2008;9:831-42. DOI: https://doi.org/10.1038/nrg2455
6. Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell 2012;148:1172-87. DOI: https://doi.org/10.1016/j.cell.2012.02.005
7. Maroney PA, Yu Y, Fisher J, Nilsen TW. Evidence that microRNAs are associated with translating messenger RNAs in human cells. Nat Struct Mol Biol 2006;13:1102-7. DOI: https://doi.org/10.1038/nsmb1174
8. Petersen CP, Bordeleau ME, Pelletier J, Sharp PA. Short RNAs repress translation after initiation in mammalian cells. Mol Cell 2006;21:533-42. DOI: https://doi.org/10.1016/j.molcel.2006.01.031
9. Hausser J, Syed AP, Bilen B, Zavolan M. Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation. Genome Res 2013;23:604-15. DOI: https://doi.org/10.1101/gr.139758.112
10. Baek D, Villen J, Shin C, et al. The impact of microRNAs on protein output. Nature 2008;455:64-71. DOI: https://doi.org/10.1038/nature07242
11. Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 2011;12:99-110. DOI: https://doi.org/10.1038/nrg2936
12. Place RF, Li LC, Pookot D, et al. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A 2008;105:1608-13. DOI: https://doi.org/10.1073/pnas.0707594105
13. Zhang X, Zuo X, Yang B, et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 2014;158:607-19. DOI: https://doi.org/10.1016/j.cell.2014.05.047
14. Li X, Pritykin Y, Concepcion CP, et al. High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down. Mol Cell 2020;79:167-79 e11. DOI: https://doi.org/10.1016/j.molcel.2020.05.009
15. Helwak A, Kudla G, Dudnakova T, Tollervey D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 2013;153:654-65. DOI: https://doi.org/10.1016/j.cell.2013.03.043
16. Hafner M, Landthaler M, Burger L, et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 2010;141:129-41. DOI: https://doi.org/10.1016/j.cell.2010.03.009
17. Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 2010;466:835-40. DOI: https://doi.org/10.1038/nature09267
18. Selbach M, Schwanhausser B, Thierfelder N, et al. Widespread changes in protein synthesis induced by microRNAs. Nature 2008;455:58-63. DOI: https://doi.org/10.1038/nature07228
19. Broughton JP, Lovci MT, Huang JL, et al. Pairing beyond the Seed Supports MicroRNA Targeting Specificity. Mol Cell 2016;64:320-33. DOI: https://doi.org/10.1016/j.molcel.2016.09.004
20. Kim JH, Lee SR, Li LH, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 2011;6:e18556. DOI: https://doi.org/10.1371/journal.pone.0018556
21. Zhang K, Zhang X, Cai Z, et al. A novel class of microRNA-recognition elements that function only within open reading frames. Nat Struct Mol Biol 2018;25:1019-27. DOI: https://doi.org/10.1038/s41594-018-0136-3
CRediT authorship contribution
Dandan Wang conceived and designed the methodology and data analysis, and reviewed and edited the manuscript.; Le Bai performed the experiments, analyzed the data and prepared figures; Le Bai and Dandan Wang analyzed the data and wrote the manuscript.
How to Cite
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
PAGEPress has chosen to apply the Creative Commons Attribution NonCommercial 4.0 International License (CC BY-NC 4.0) to all manuscripts to be published.