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Here, we present a programmable strategy to enhance IRES-dependent translation by recruiting a truncated eIF4G scaffold via engineered RNA-binding protein fusions. A \u03bbN\u2013eIF4G fusion selectively increased EMCV IRES-mediated translation without affecting cap-dependent expression, achieving up to 7.7-fold enhancement. Translation output was tunable over a broad dynamic range through effector dosage and linker design, revealing key design parameters for controlling activity. The system functioned across multiple mammalian cell lines and in tricistronic constructs, enabling coordinated regulation of polycistronic expression. Computational modeling was consistent with preferential interaction between \u03bbN and the EMCV IRES. These results establish a modular framework for the programmable control of translation, define design principles for RNA-guided translational control, and expand the synthetic biology toolkit for post-transcriptional gene regulation.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00420","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T04:30:13Z","timestamp":1788323413000},"page":"2905-2915","source":"Crossref","is-referenced-by-count":0,"title":["Programmable\nEnhancement of IRES-Mediated Translation\nvia RNA-Guided eIF4G Recruitment"],"prefix":"10.1021","volume":"65","author":[{"given":"Binbin","family":"Ying","sequence":"first","affiliation":[{"name":"Kyushu University , , 744 Motooka, Nishi-ku , ,","place":["Fukuoka, Japan, 819-0395"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feiyang","family":"Zheng","sequence":"additional","affiliation":[{"name":"Kyushu University , , 744 Motooka, Nishi-ku , ,","place":["Fukuoka, Japan, 819-0395"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshinori","family":"Kawabe","sequence":"additional","affiliation":[{"name":"Kyushu University , , 744 Motooka, Nishi-ku , ,","place":["Fukuoka, Japan, 819-0395"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2570-5712","authenticated-orcid":true,"given":"Masamichi","family":"Kamihira","sequence":"additional","affiliation":[{"name":"Kyushu University , , 744 Motooka, Nishi-ku , ,","place":["Fukuoka, Japan, 819-0395"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"2026091508070729900_cit1","doi-asserted-by":"publisher","first-page":"2636","DOI":"10.1128\/jvi.62.8.2636-2643.1988","article-title":"A Segment of the 5\u2032 Nontranslated Region of\nEncephalomyocarditis Virus RNA Directs Internal Entry of Ribosomes\nduring In Vitro Translation","volume":"62","author":"Jang","year":"1988","journal-title":"J. Virol."},{"key":"2026091508070729900_cit2","doi-asserted-by":"publisher","first-page":"320","DOI":"10.1038\/334320a0","article-title":"Internal Initiation\nof Translation of Eukaryotic mRNA\nDirected by a Sequence Derived from Poliovirus RNA","volume":"334","author":"Pelletier","year":"1988","journal-title":"Nature"},{"key":"2026091508070729900_cit3","doi-asserted-by":"publisher","first-page":"882","DOI":"10.1016\/j.tibs.2018.04.011","article-title":"Cap-Independent\nTranslation: What\u2019s in a Name?","volume":"43","author":"Shatsky","year":"2018","journal-title":"Trends\nBiochem. Sci."},{"key":"2026091508070729900_cit4","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0082100","article-title":"An Internal\nRibosome Entry Site Mutant Library for Tuning Expression\nLevel of Multiple Genes in Mammalian Cells","volume":"8","author":"Koh","year":"2013","journal-title":"PLoS One"},{"key":"2026091508070729900_cit5","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1016\/S1389-1723(04)00285-3","article-title":"Production of Anti-CD2 Chimeric Antibody\nby Recombinant\nAnimal Cells","volume":"98","author":"Hotta","year":"2004","journal-title":"J. Biosci. Bioeng."},{"key":"2026091508070729900_cit6","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1093\/protein\/gzm038","article-title":"Analysis of\nIgG heavy chain to light chain ratio with mutant Encephalomyocarditis\nvirus internal ribosome entry site","volume":"20","author":"Li","year":"2007","journal-title":"Protein\nEng. Des. Sel."},{"key":"2026091508070729900_cit7","doi-asserted-by":"publisher","first-page":"130","DOI":"10.1016\/j.jbiotec.2011.09.023","article-title":"IRES-Mediated Tricistronic Vectors\nfor Enhancing Generation of High Monoclonal Antibody Expressing CHO\nCell Lines","volume":"157","author":"Ho","year":"2012","journal-title":"J. Biotechnol."},{"key":"2026091508070729900_cit8","doi-asserted-by":"publisher","first-page":"11","DOI":"10.5493\/wjem.v5.i1.11","article-title":"Internal Ribosome\nEntry Site-Based Vectors for Combined\nGene Therapy","volume":"5","author":"Renaud-Gabardos","year":"2015","journal-title":"World J. Exp. Med."},{"key":"2026091508070729900_cit9","doi-asserted-by":"publisher","DOI":"10.3390\/biomedicines10081865","article-title":"Internal Ribosome\nEntry Site (IRES)-Mediated\nTranslation and Its Potential for Novel mRNA-Based Therapy Development","volume":"10","author":"Marques","year":"2022","journal-title":"Biomedicines"},{"key":"2026091508070729900_cit10","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkm1119","article-title":"Improvement of Reporter Activity\nby IRES-Mediated Polycistronic Reporter System","volume":"36","author":"Bouabe","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"2026091508070729900_cit11","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac1252","article-title":"Synthetic Circular RNA Switches and\nCircuits That Control Protein Expression in Mammalian Cells","volume":"51","author":"Kameda","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"2026091508070729900_cit12","doi-asserted-by":"publisher","first-page":"1656","DOI":"10.1021\/acssynbio.7b00124","article-title":"Artificial OFF-Riboswitches That\nDownregulate Internal Ribosome Entry without Hybridization Switches\nin a Eukaryotic Cell-Free Translation System","volume":"6","author":"Ogawa","year":"2017","journal-title":"ACS Synth. Biol."},{"key":"2026091508070729900_cit13","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines11020238","article-title":"Cap-Independent Circular\nmRNA Translation Efficiency","volume":"11","author":"Deviatkin","year":"2023","journal-title":"Vaccines"},{"key":"2026091508070729900_cit14","doi-asserted-by":"publisher","first-page":"127","DOI":"10.4014\/jmb.1811.11019","article-title":"Development of an RNA Expression\nPlatform Controlled\nby Viral Internal Ribosome Entry Sites","volume":"29","author":"Ko","year":"2019","journal-title":"J. Microbiol.\nBiotechnol."},{"key":"2026091508070729900_cit15","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22105193","article-title":"Current Practice in Bicistronic IRES Reporter Use:\nA Systematic Review","volume":"22","author":"van den Akker","year":"2021","journal-title":"Int. J. Mol. 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Bacteriol."},{"key":"2026091508070729900_cit31","doi-asserted-by":"publisher","first-page":"4138","DOI":"10.1093\/nar\/gkf552","article-title":"RNA Recognition Site of PP7 Coat Protein","volume":"30","author":"Lim","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"2026091508070729900_cit32","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a033092","article-title":"Protein Synthesis Initiation in Eukaryotic Cells","volume":"10","author":"Merrick","year":"2018","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"2026091508070729900_cit33","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1038\/nrm2838","article-title":"The Mechanism\nof Eukaryotic Translation\nInitiation and Principles of Its Regulation","volume":"11","author":"Jackson","year":"2010","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2026091508070729900_cit34","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-50776-z","article-title":"Translational\nenhancement of target endogenous mRNA in mammalian cells using programmable\nRNA-binding pentatricopeptide repeat proteins","volume":"14","author":"Ping","year":"2024","journal-title":"Sci. Rep."},{"key":"2026091508070729900_cit35","doi-asserted-by":"publisher","first-page":"4865","DOI":"10.1093\/emboj\/18.17.4865","article-title":"Translation driven\nby an eIF4G core domain in vivo","volume":"18","author":"De Gregorio","year":"1999","journal-title":"EMBO J."},{"key":"2026091508070729900_cit36","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction\nof biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"2026091508070729900_cit37","doi-asserted-by":"publisher","first-page":"117","DOI":"10.1038\/s41592-023-02086-5","article-title":"Accurate prediction of protein-nucleic acid complexes\nusing RoseTTAFoldNA","volume":"21","author":"Baek","year":"2024","journal-title":"Nat. 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