Site-Specific Inhibition of Translation Initiation via 2’-O-methylation
Posted on: 17 June 2026
Preprint posted on 20 January 2026
Categories: biochemistry, cell biology, genetics
Background:
The translation of RNA into functional proteins is an important part of the central dogma of molecular biology. Translation can be divided into three fundamental processes: initiation of translation, elongation, and finally termination through the release of the formed polypeptide chain.
In eukaryotes, the initiation region of translation is marked by the so-called Korzak consensus sequence, which acts as the initiation site for translation. The Korzak sequence describes a specific set of nucleotides located around the start codon, most commonly GCCRCC AUG G, where R can be either adenosine or guanosine [1]. Translation can also be initiated by either start codons, that are not embedded into a Korzak sequence or near-cognate start codons (e.g. CUG, GUG or UUG), which can lead to translation of so called upstream open reading frames (uORFs), associated with regulation of translation as well as the formation of micropeptides, often associated with a variety of different diseases [2,3].
In this paper, the authors examine the function of the epitranscriptome specifically in relation to the initiation of translation. The epitranscriptom describes the modification of mRNA, which are dynamic and reversible and relate to genome stability, splicing and or translation [4]. Despite extensive studies of the epitranscriptome, particularly the modifications m6A, Ψ, m5C, and A-to-I editing, many properties and effects of the epitranscriptome remain unclear.
Therefore, the authors focus on the effects of 2′-O-methylation of the nucleotides of the Korzak sequence. This involves the substitution of the 2′-OH group of ribose with a methyl group. Through this substitution, the authors were able to demonstrate an inhibitory effect of the 2′-O-methylation by failed initiation complex formation due to missed start codon recognition. The start codon remains unrecognized due to the 2′-O-methylation and missing H-bond to the corresponding 18srRNA. It is interesting to note that 2′-O-methylation has inhibitory functions at the upstream start codon, while subsequently demonstrating an enhancing effect on the translation of the downstream ORFs.
Results:
2’-O-methyl modification at the A[+1] Position of AUG inhibits protein synthesis
The authors systematically introduced 2′-O-methylation into the Kozak sequence using both three-way and two-way splint ligation approaches. In the three-way approach, a 5′UTR lacking a start codon, a NanoLuc ORF without a start codon, and a synthetic Kozak sequence with or without 2′-O-methylation were ligated. The two-way approach instead ligated a modified 5′UTR/Kozak sequence to the NanoLuc ORF, but its use was limited by the need for chemical capping and size restrictions. These experiments validated the three-way method and confirmed that the observed effects were not due to background noise or unligated material.
Testing the constructs in HeLa cells showed that 2′-O-methylation at the first nucleotide of the AUG start codon strongly inhibits protein synthesis. Modifications at the G position of AUG or the following G also reduced expression, whereas modifications upstream of the start codon had no effect. Replacing NanoLuc with GFP reproduced these findings. Based on these results, the authors concluded that the 2′OH group of the first nucleotide in the start codon is required for interaction with U1830 of the 18S rRNA during translation initiation.
Preprint Figure 1: Schematic representation of the hydrogen bonding network during the preinitiation complex. Particular attention is paid to the hydrogen bond between the first position of the start codon A[+1] and U1830 of the 18S rRNA.
2′-O-methylation displays the greatest inhibitory effect at cognate AUG start codons
The authors referred to a previous study identifying alternative translation initiation sites, where CUG, AUG, and GUG were the most frequently used start codons. This raised the question of whether 2′-O-methylation also inhibits translation initiation at near-cognate start codons. To test this, they examined different codon variants and additionally assessed whether the effect was adenine-specific by retaining A at codon position 1 while altering the nucleotides at positions 2 and 3. Their results showed that 2′-O-methylation has the strongest inhibitory effect on the cognate AUG start codon.
2’O Modification inhibits startcodon recognition by the ribosome
By inserting a uORF with either a modified or unmodified start codon, the authors showed that Am[+1] reduces recognition of upstream start codons and increases initiation at downstream start codons. To test whether 2′O-modification causes ribosomal stalling, they used GMPPNP to stall the 48S pre-initiation complex and harringtonine to stall the 80S complex before elongation. The results showed that neither complex formed efficiently, indicating that the defect occurs during start codon recognition rather than ribosome stalling.
2′-O-methylation naturally occurs in near-cognate startcodons and acts as an inhibitor
The authors provide evidence that Nm[+1] modifications are enriched in upstream near-cognate start codons within 5′UTRs, whereas canonical AmUG sites appear absent or extremely rare in HeLa and C4-2 cells. Using RTL-P and FBL knockdown, they validate several Nm-modified near-cognate codons and show that loss of these modifications reduces translation efficiency and canonical protein expression for different targets. While the transcriptome-wide effects are modest, likely due to low Nm stoichiometry at many sites, the data support a regulatory role for 2′-O-methylation in modulating uORF usage and downstream translation.
Why I highlight this work:
Protein translation describes one of the most important core processes of molecular biology. When viewed in its entirety, this process is characterized by numerous regulatory modules that determine both its precision as well as its adaptability. One of the least studied modules is the epitranscriptome, which describes the modification of mRNA and its downstream effects on translation. In this paper, the authors systematically investigate Nm modification using a variety of methods and a toolkit provided by nature itself.
From a thematic perspective, the paper addresses a niche topic whose physiological relevance is by no means overshadowed by the analyses presented herein. The paper stands out for its rigorous experimental design and a multitude of controls that consistently support the experiments’ findings. These further substantiate the results and always seem to answer the reader’s next question as it forms in their mind.
Overall, the paper not only presents what I personally find to be a very charming and experimentally sound approach to the production of chimeric RNA, but also highlights the beauty of the fine-tuning of our own organism, its complexity, and its fragility in terms of protein translation.
Lastly, the therapeutic benefits of the work presented here should not be forgotten. The results presented provide a solid basis for therapeutic measures to prevent unwanted 5’UTR translation in the fight against disease.
Of course, I approach this paper with a certain bias, as I myself come from the field of ribosome research. Even though I originally read the paper for the methodology section, I really enjoyed the results section and was able to broaden my understanding of such a fundamental and essential process within my own biology.
References
[1] Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125-48. doi: 10.1093/nar/15.20.8125. PMID: 3313277; PMCID: PMC306349.
[2] Calvo, S.E., Pagliarini, D.J. and Mootha, V.K. (2009) Upstream open reading frames 802 cause widespread reduction of protein expression and are polymorphic among 803 humans. Proc Natl Acad Sci U S A, 106, 7507-7512.
[3] Dasgupta, A. and Prensner, J.R. (2024) Upstream open reading frames: new players 805 in the landscape of cancer gene regulation. NAR Cancer, 6, zcae023.
[4] Ruden DM. Uncovering the Epitranscriptome: A Review on mRNA Modifications and Emerging Frontiers. Genes (Basel). 2025 Aug 12;16(8):951. doi: 10.3390/genes16080951. PMID: 40870000; PMCID: PMC12385769.
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| List by | Theodora Stougiannou et al. |
BSDB Spring Meeting: Molecules to Morphogenesis
The British Society for Developmental Biology (BSDB) Spring Meeting Molecules to Morphogenesis was held from 23–26 March 2026 at the University of Warwick (UK). This meeting brought together a vibrant community of researchers to discuss how molecular mechanisms are integrated across scales to drive morphogenesis, spanning diverse model systems and approaches. This preList contains preprints by presenters from the talk and poster sessions at the meeting. Please do get in touch at preLights@biologists.com if you notice any relevant preprints that we may have missed.
| List by | Ingrid Tsang |
Keystone Symposium on Stem Cell Models in Embryology 2026
The Keystone Symposium on Stem Cell Models in Embryology, 2026, was organised by Jun Wu (UT Southwestern), Jianping Fu (University of Michigan) and Miki Ebisuya (TU Dresden) and held at Asilomar Conference Grounds in California (US). The meeting discussed recent advances made in establishing stem-cell-based embryo models, what fundamental insights into developmental processes have been gleaned from them, as well as how they are beginning to be applied more widely. This prelist contains preprints by presenters at the talk and poster sessions at the conference, which our Reviews Editor in attendance spotted. Please do reach out to preLights@biologists.com if you notice any that we’ve missed.
| List by | Ingrid Tsang |
SciELO preprints – From 2025 onwards
SciELO has become a cornerstone of open, multilingual scholarly communication across Latin America. Its preprint server, SciELO preprints, is expanding the global reach of preprinted research from the region (for more information, see our interview with Carolina Tanigushi). This preList brings together biological, English language SciELO preprints to help readers discover emerging work from the Global South. By highlighting these preprints in one place, we aim to support visibility, encourage early feedback, and showcase the vibrant research communities contributing to SciELO’s open science ecosystem.
| List by | Carolina Tanigushi |
October in preprints – DevBio & Stem cell biology
Each month, preLighters with expertise across developmental and stem cell biology nominate a few recent developmental and stem cell biology (and related) preprints they’re excited about and explain in a single paragraph why. Short, snappy picks from working scientists — a quick way to spot fresh ideas, bold methods and papers worth reading in full. These preprints can all be found in the October preprint list published on the Node.
| List by | Deevitha Balasubramanian et al. |
September in preprints – Cell biology edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading list. This month, categories include: (1) Cell organelles and organisation, (2) Cell signalling and mechanosensing, (3) Cell metabolism, (4) Cell cycle and division, (5) Cell migration
| List by | Sristilekha Nath et al. |
July in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: (1) Cell Signalling and Mechanosensing (2) Cell Cycle and Division (3) Cell Migration and Cytoskeleton (4) Cancer Biology (5) Cell Organelles and Organisation
| List by | Girish Kale et al. |
June in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: (1) Cell organelles and organisation (2) Cell signaling and mechanosensation (3) Genetics/gene expression (4) Biochemistry (5) Cytoskeleton
| List by | Barbora Knotkova et al. |
May in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) Biochemistry/metabolism 2) Cancer cell Biology 3) Cell adhesion, migration and cytoskeleton 4) Cell organelles and organisation 5) Cell signalling and 6) Genetics
| List by | Barbora Knotkova et al. |
Keystone Symposium – Metabolic and Nutritional Control of Development and Cell Fate
This preList contains preprints discussed during the Metabolic and Nutritional Control of Development and Cell Fate Keystone Symposia. This conference was organized by Lydia Finley and Ralph J. DeBerardinis and held in the Wylie Center and Tupper Manor at Endicott College, Beverly, MA, United States from May 7th to 9th 2025. This meeting marked the first in-person gathering of leading researchers exploring how metabolism influences development, including processes like cell fate, tissue patterning, and organ function, through nutrient availability and metabolic regulation. By integrating modern metabolic tools with genetic and epidemiological insights across model organisms, this event highlighted key mechanisms and identified open questions to advance the emerging field of developmental metabolism.
| List by | Virginia Savy, Martin Estermann |
April in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) biochemistry/metabolism 2) cell cycle and division 3) cell organelles and organisation 4) cell signalling and mechanosensing 5) (epi)genetics
| List by | Vibha SINGH et al. |
March in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) cancer biology 2) cell migration 3) cell organelles and organisation 4) cell signalling and mechanosensing 5) genetics and genomics 6) other
| List by | Girish Kale et al. |
Biologists @ 100 conference preList
This preList aims to capture all preprints being discussed at the Biologists @100 conference in Liverpool, UK, either as part of the poster sessions or the (flash/short/full-length) talks.
| List by | Reinier Prosee, Jonathan Townson |
Early 2025 preprints – the genetics & genomics edition
In this community-driven preList, a group of preLighters, with expertise in different areas of genetics and genomics have worked together to create this preprint reading list. Categories include: 1) bioinformatics 2) epigenetics 3) gene regulation 4) genomics 5) transcriptomics
| List by | Chee Kiang Ewe et al. |
January in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) biochemistry/metabolism 2) cell migration 3) cell organelles and organisation 4) cell signalling and mechanosensing 5) genetics/gene expression
| List by | Barbora Knotkova et al. |
End-of-year preprints – the genetics & genomics edition
In this community-driven preList, a group of preLighters, with expertise in different areas of genetics and genomics have worked together to create this preprint reading list. Categories include: 1) genomics 2) bioinformatics 3) gene regulation 4) epigenetics
| List by | Chee Kiang Ewe et al. |
BSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
| List by | Joyce Yu, Katherine Brown |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
| List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
| List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
| List by | Shikha Nayar |