January 19th, 2025

Recent Publications Harnessing the Power of Translatomics

Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.

In this week’s Sunday papers:

  • Tzani et al. use ribosome profiling to highlight the significance of microproteins and non-canonical translation in CHO cells.
  • Zhang et al. use ribosome profiling and polysome profiling to explore the metabolic regulation of satellite cell activation by identifying metabolites that facilitate this process.
  • Inoue et al. re-analyse Ribo-Seq and RNA-Seq to examine the factors influencing shape preference behavior in medaka fish.

Detection of host cell microprotein impurities in antibody drug products

Nature Communications, 2024

Tzani, I., Castro-Rivadeneyra, M., Kelly, P., Strasser, L., Zhang, L., Clynes, M., Karger, B.L., Barron, N., Bones, J. and Clarke, C.

Chinese hamster ovary (CHO) cells are critical for producing nearly 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). However, gaps in the annotation of non-canonical translation events in these cellular systems hinder deeper exploration of CHO cell biology and the identification of host cell protein (HCP) impurities in antibody drug products. HCPs in the final drug product are a significant concern as they may trigger an immune response in patients or diminish the drug’s efficacy. Regulatory authorities recommend using mass spectrometry (MS) for HCP detection. MS enables the identification and quantification of individual HCPs, including those present at low concentrations. Despite significant advancements in transcriptome annotation, proteome characterization remains more challenging and incomplete. This limitation restricts the ability of MS to comprehensively detect the full spectrum of potential HCP impurities.

To address this, the authors employed ribosome footprint profiling (Ribo-Seq) with translational inhibitors harringtonine and cycloheximide to uncover novel open reading frames (ORFs), including N-terminal extensions and thousands of short ORFs (sORFs) that potentially encode microproteins. Using an expanded protein sequence database, mass spectrometry-based HCP analysis of eight commercial antibody drug products (seven mAbs and one Fc-fusion protein) confirmed the presence of microprotein impurities. Less than 5% of predicted with Ribo-seq microproteins (173/3681) were confirmed with mass spectrometry. Their findings also reveal that microprotein levels vary depending on the growth phase and are influenced by temperature reduction. This study not only highlights the significance of non-canonical translation in CHO cells but also provides a valuable resource for advancing research on protein synthesis regulation and improving the understanding of impurities in therapeutic antibody production. The results offer crucial insights for enhancing the quality and safety of antibody-based therapeutics.

Learn more about EIRNA Bio’s ribosome profiling service here.

Spermidine-eIF5A axis is essential for muscle stem cell activation via translational control

Nature Cell Discovery, 2024

Zhang, Q., Han, W., Wu, R., Deng, S., Meng, J., Yang, Y., Li, L., Sun, M., Ai, H., Chen, Y., Liu, Q. et al.

Adult skeletal muscle stem cells, known as satellite cells (SCs), remain in a quiescent state but activate in response to injury, re-enter the cell cycle, and subsequently differentiate and fuse to facilitate muscle repair. As we age, muscle mass and strength gradually diminish, largely due to impaired activation and reduced self-renewal of SCs. Understanding the molecular mechanisms underlying SC activation could open new pathways for treating muscle-degenerative diseases and combating age-related muscle decline. In this study, the authors explored the metabolic regulation of SC activation by identifying metabolites that facilitate this process.

Through targeted metabolomics and use of single-cell RNA-Seq, ribosome- and polysome profiling, they discovered that spermidine serves as a key regulatory metabolite, promoting SC activation and muscle regeneration in mice. Mechanistically, spermidine drives SC activation by inducing the generation of hypusinated eIF5A. MyoD is a myogenic lineage-specific transcription factor that has long been used as a molecular marker for SC activation. Using SC-specific eIF5A-knockout (KO) and Myod-KO mice, they demonstrated that eIF5A is essential for spermidine-mediated SC activation by regulating MyoD translation. Importantly, the loss of eIF5A in SCs significantly impairs muscle regeneration in mice. Collectively, these findings reveal a novel mechanism critical for SC activation, operating through the spermidine-eIF5A axis to regulate MyoD translation. This axis represents a promising pharmacological target for activating endogenous SCs to treat muscular diseases.

Learn more about EIRNA Bio’s ribosome profiling and polysome profiling services here.

An evolutionarily distinct Hmgn2 variant influences shape recognition in Medaka Fish

Nature Communications Biology, 2024

Inoue, S., Masaki, Y., Nakagawa, S. and Yokoi, S.

A dominant theory in molecular biology posits that diversification of species largely stems from alterations in cis-regulatory elements such as enhancers, silencers, and insulators. These modifications can drive differential gene expression patterns, even when protein-coding sequences remain conserved, thereby contributing to phenotypic variation. In addition to cis-regulatory changes, the evolution of novel proteins with unique amino acid sequences may also play a crucial role in driving evolutionary processes. Shape perception, a vital cognitive function for the survival of many species, allows organisms to navigate complex environments, locate resources, evade predators, and interact socially. While the mechanisms underlying shape perception are well-studied in mammals, they remain less understood in non-mammalian species.

In this study, the authors identified a novel protein variant influencing shape preference behavior in medaka fish (Oryzias latipes). Re-analysis of Ribo-Seq and RNA-Seq sequencing data revealed that LOC101156433 encodes a distinct Hmgn2 gene variant with atypical subnuclear localization, clustering separately from Hmgn2 clades in other species. Knockout Medaka lines carrying this Hmgn2 variant with 2bp deletion exhibited reduced telencephalic regions and altered shape preference behavior, suggesting a connection between protein sequence variation and behavioral changes. Notably, this Hmgn2 variant is prevalent among Acanthopterygii fishes, a group adapted to diverse environments, pointing to its potential evolutionary significance. These findings underscore the impact of amino acid sequence variation on the emergence of novel molecular and behavioral adaptations, offering new insights into the visual shape perception system in fish.

Learn more about EIRNA Bio’s ribosome-seq service here.

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