{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:06:03Z","timestamp":1789391163272,"version":"build-2803163510"},"reference-count":41,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"publisher","award":["075-15-2025-470"],"award-info":[{"award-number":["075-15-2025-470"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Internal ribosome entry site (IRES) elements have been proposed for constructing polycistronic mRNAs in Komagataella phaffii, but their reported activity is inconsistent and has not been evaluated under standardized conditions. Here, a standardized bicistronic EGFP-IRES-mKate2 system was developed in which the cap-dependent first cistron (EGFP) reports promoter activity, and translation of the second (mKate2) should depend on the IRES. The RhPV, TEV, PVY, and Saccharomyces cerevisiae GPR1 elements were assessed under PAOX1 and PTHI11, with EMCV and HCV as negative controls. EGFP tracked promoter induction and repression, whereas mKate2 remained at background for GPR1, EMCV, and HCV; for RhPV, TEV, and PVY it was low, promoter-independent, and persisted after promoter deletion. By RT-qPCR, the transcript was intact across both cistrons, except in the RhPV construct, where its 3\u2032 region was depleted. The residual synthesis therefore reflected cryptic promoter activity below 1% of that of PAOX1, not IRES-dependent initiation. None of the elements directed detectable cap-independent translation above 0.4% of cap-dependent expression of the same ORF, limiting their use for polycistronic expression. The system, the first to place IRES activity in K. phaffii on a quantitative scale, distinguishes IRES-dependent translation from cryptic transcription and can facilitate the search for functional elements.<\/jats:p>","DOI":"10.3390\/ijms27188166","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T12:09:51Z","timestamp":1789387791000},"page":"8166","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Reassessment of IRES Element Activity in Komagataella phaffii Using a Standardized Bicistronic System: Cryptic Promoter Activity Mimics Cap-Independent Translation"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-6323-7156","authenticated-orcid":false,"given":"Danil S.","family":"Kalinin","sequence":"first","affiliation":[{"name":"A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Ekaterina V.","family":"Schetinina","sequence":"additional","affiliation":[{"name":"A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Alexey N.","family":"Fedorov","sequence":"additional","affiliation":[{"name":"A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1368-2146","authenticated-orcid":false,"given":"Igor E.","family":"Granovsky","sequence":"additional","affiliation":[{"name":"A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia"},{"name":"Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences, Pushchino 142290, Moscow Region,   Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1431","DOI":"10.1007\/s00018-016-2409-5","article-title":"A Researcher\u2019s Guide to the Galaxy of IRESs","volume":"74","author":"Terenin","year":"2016","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1093\/jmcb\/mjz091","article-title":"IRES-Mediated Cap-Independent Translation, a Path Leading to Hidden Proteome","volume":"11","author":"Yang","year":"2019","journal-title":"J. Mol. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1093\/emboj\/cdg103","article-title":"Internal Initiation Drives the Synthesis of Ure2 Protein Lacking the Prion Domain and Affects [URE3] Propagation in Yeast Cells","volume":"22","author":"Komar","year":"2003","journal-title":"EMBO J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.bbaexp.2004.11.009","article-title":"Translational Control by Internal Ribosome Entry Site in Saccharomyces cerevisiae","volume":"1681","author":"Seino","year":"2005","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1007\/s10529-012-0862-2","article-title":"Internal Ribosome Entry Site Mediates Protein Synthesis in Yeast Pichia pastoris","volume":"34","author":"Liang","year":"2012","journal-title":"Biotechnol. Lett."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1186\/s13068-019-1640-3","article-title":"Screening for Functional IRESes Using \u03b1-Complementation System of \u03b2-Galactosidase in Pichia pastoris","volume":"12","author":"Huang","year":"2019","journal-title":"Biotechnol. Biofuels"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5867","DOI":"10.1002\/jcp.29583","article-title":"Pichia pastoris: A Highly Successful Expression System for Optimal Synthesis of Heterologous Proteins","volume":"235","author":"Karbalaei","year":"2020","journal-title":"J. Cell. Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Khlebodarova, T.M., Bogacheva, N.V., Zadorozhny, A.V., Bryanskaya, A.V., Vasilieva, A.R., Chesnokov, D.O., Pavlova, E.I., and Peltek, S.E. (2024). Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry. Microorganisms, 12.","DOI":"10.3390\/microorganisms12020346"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"51615","DOI":"10.1039\/C4RA07679F","article-title":"Development of a Prokaryotic-like Polycistronic Expression System Based on a Virus-Originated Internal Ribosome Entry Site (IRES) in Industrial Eukaryotic Microorganisms","volume":"4","author":"Xu","year":"2014","journal-title":"RSC Adv."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4733","DOI":"10.1073\/pnas.0710650105","article-title":"Splicing Mediates the Activity of Four Putative Cellular Internal Ribosome Entry Sites","volume":"105","author":"Baranick","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6593","DOI":"10.1093\/nar\/gki958","article-title":"A Second Look at Cellular MRNA Sequences Said to Function as Internal Ribosome Entry Sites","volume":"33","author":"Kozak","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e1458","DOI":"10.1002\/wrna.1458","article-title":"Viral Internal Ribosomal Entry Sites: Four Classes for One Goal","volume":"9","author":"Mailliot","year":"2018","journal-title":"WIREs RNA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12141","DOI":"10.1128\/JVI.75.24.12141-12152.2001","article-title":"Cap-Independent Translation Conferred by the 5\u2032 Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent","volume":"75","author":"Gallie","year":"2001","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"26813","DOI":"10.1074\/jbc.M503576200","article-title":"Cap-Independent Translation of Tobacco Etch Virus Is Conferred by an RNA Pseudoknot in the 5\u2032-Leader","volume":"280","author":"Zeenko","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2131","DOI":"10.1271\/bbb.61.2131","article-title":"Mutational Analysis of the Potato Virus Y 5\u2032 Untranslated Region for Alteration in Translational Enhancement in Tobacco Protoplasts","volume":"61","author":"Yang","year":"1997","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1099\/vir.0.18781-0","article-title":"In Vivo Activity of Rhopalosiphum padi Virus Internal Ribosome Entry Sites","volume":"84","author":"Domier","year":"2003","journal-title":"J. Gen. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2753","DOI":"10.1101\/gad.1832209","article-title":"RPS25 Is Essential for Translation Initiation by the Dicistroviridae and Hepatitis C Viral IRESs","volume":"23","author":"Landry","year":"2009","journal-title":"Genes Dev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1347","DOI":"10.1002\/j.1460-2075.1990.tb08249.x","article-title":"Expression of Potyviral Polyproteins in Transgenic Plants Reveals Three Proteolytic Activities Required for Complete Processing","volume":"9","author":"Carrington","year":"1990","journal-title":"EMBO J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/0378-1119(95)00521-7","article-title":"The Tobacco Etch Viral 5\u2032 Leader and Poly(A) Tail Are Functionally Synergistic Regulators of Translation","volume":"165","author":"Gallie","year":"1995","journal-title":"Gene"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1565","DOI":"10.1099\/vir.0.79992-0","article-title":"The Rhopalosiphum padi Virus 5\u2032 Internal Ribosome Entry Site Is Functional in Spodoptera Frugiperda 21 Cells and in Their Cell-Free Lysates: Implications for the Baculovirus Expression System","volume":"85","author":"Royall","year":"2004","journal-title":"J. Gen. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/BF01703392","article-title":"The 5\u2032 Untranslated Region of PVY RNA, Even Located in an Internal Position, Enables Initiation of Translation","volume":"7","author":"Levis","year":"1993","journal-title":"Virus Genes"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.virusres.2015.02.005","article-title":"The Role of the 5\u2032 Untranslated Regions of Potyviridae in Translation","volume":"206","author":"Zhang","year":"2015","journal-title":"Virus Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4990","DOI":"10.1128\/MCB.20.14.4990-4999.2000","article-title":"Naturally Occurring Dicistronic Cricket Paralysis Virus RNA Is Regulated by Two Internal Ribosome Entry Sites","volume":"20","author":"Wilson","year":"2000","journal-title":"Mol. Cell. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1016\/S0022-2836(02)01099-9","article-title":"Factorless Ribosome Assembly on the Internal Ribosome Entry Site of Cricket Paralysis Virus","volume":"324","author":"Jan","year":"2002","journal-title":"J. Mol. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1016\/j.virol.2011.01.005","article-title":"Mechanism of Translation Initiation by Dicistroviridae IGR IRESs","volume":"411","author":"Hertz","year":"2011","journal-title":"Virology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10244","DOI":"10.1128\/JVI.75.21.10244-10249.2001","article-title":"The 5\u2032 Untranslated Region of Rhopalosiphum padi Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems","volume":"75","author":"Woolaway","year":"2001","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1006\/viro.1995.0081","article-title":"Cis-Acting Elements of the Encephalomyocarditis Virus Internal Ribosomal Entry Site","volume":"214","author":"Witherell","year":"1995","journal-title":"Virology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"283","DOI":"10.2144\/000112243","article-title":"Translational Efficiency of EMCV IRES in Bicistronic Vectors Is Dependent Upon IRES Sequence and Gene Location","volume":"41","author":"Bochkov","year":"2006","journal-title":"Biotechniques"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10827","DOI":"10.1128\/JVI.74.22.10827-10833.2000","article-title":"Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5\u2032 Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type","volume":"74","author":"Laporte","year":"2000","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"596","DOI":"10.4161\/rna.7.5.13096","article-title":"Components of the Multifactor Complex Needed for Internal Initiation by the IRES of Hepatitis C Virus in Saccharomyces cerevisiae","volume":"7","author":"Rosenfeld","year":"2010","journal-title":"RNA Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1449","DOI":"10.1111\/j.1478-3231.2011.02543.x","article-title":"Hepatitis C Viral Protein Translation: Mechanisms and Implications in Developing Antivirals","volume":"31","author":"Hoffman","year":"2011","journal-title":"Liver Int."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1224","DOI":"10.1126\/science.1144467","article-title":"Cap-Independent Translation Is Required for Starvation-Induced Differentiation in Yeast","volume":"317","author":"Gilbert","year":"2007","journal-title":"Science"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1016\/j.jbiotec.2010.09.957","article-title":"Identification and Characterisation of Novel Pichia Pastoris Promoters for Heterologous Protein Production","volume":"150","author":"Stadlmayr","year":"2010","journal-title":"J. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.nbt.2012.11.010","article-title":"Regulation of Pichia Pastoris Promoters and Its Consequences for Protein Production","volume":"30","author":"Vogl","year":"2013","journal-title":"New Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: Pairwise Alignment for Nucleotide Sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"21","DOI":"10.4103\/2229-5186.79345","article-title":"Methods for the Determination of Limit of Detection and Limit of Quantitation of the Analytical Methods","volume":"2","author":"Shrivastava","year":"2011","journal-title":"Chron. Young Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2672","DOI":"10.1021\/ac981179n","article-title":"Experimental Comparison of the Different Approaches to Estimate LOD and LOQ of an HPLC Method","volume":"71","author":"Vial","year":"1999","journal-title":"Anal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (2002). Immunoblotting and Immunodetection. Short Protocols in Molecular Biology, John Wiley & Sons. [5th ed.]. Unit 10.8.","DOI":"10.1002\/0471142727"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"115037","DOI":"10.1016\/j.jviromet.2024.115037","article-title":"A Method for Producing Protease pS273R of the African Swine Fever Virus","volume":"330","author":"Kalinin","year":"2024","journal-title":"J. Virol. Methods"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1017\/S1355838299981682","article-title":"Genome-Wide Bioinformatic and Molecular Analysis of Introns in Saccharomyces cerevisiae","volume":"5","author":"Spingola","year":"1999","journal-title":"RNA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"14055","DOI":"10.1073\/pnas.96.24.14055","article-title":"In Silico Detection of Control Signals: mRNA 3\u2032-End-Processing Sequences in Diverse Species","volume":"96","author":"Graber","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8166\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T12:28:30Z","timestamp":1789388910000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8166"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,14]]},"references-count":41,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ijms27188166"],"URL":"https:\/\/doi.org\/10.3390\/ijms27188166","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,14]]}}}