June 8th, 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,

  • Fuchs et al. show that ribosome profiling variably detects conventional and cryptic T-cell antigen sources, highlighting its strengths and limitations in immunotherapy discovery.
  • Kore et al. uncover thousands of small open reading frame-encoded proteins (SEPs) in the human genome, some linked to disease and immune presentation, expanding our view of the proteome.
  • Finally, Halasz et al. identify the lncRNA LOUP as a multifunctional regulator of macrophage differentiation and inflammation, acting through both gene regulation and microprotein coding.

Together, these studies deepen our understanding of hidden translon potential and its role in immunity and disease.

Ribosome profiling shows variable sensitivity to detect open reading frames for conventional and different types of cryptic T-cell antigens

Molecular Therapy, Methods & Clinical Development, 2025

Fuchs K.J., Thomaidou S., van der Slik A.R., van de Meent M., ‘t Hoen P., Falkenburg J.F., Zaldumbide A., Griffioen M.

The paper investigates the effectiveness of ribosome profiling (Ribo-seq) in identifying various open reading frames (ORFs) that encode T-cell antigens, including both conventional and cryptic types. Effective T-cell-based immunotherapies targeting minor histocompatibility antigens (MiHAs) by means of allogeneic stem cell transplantation, rely on accurately identifying these antigenic peptides. While conventional antigens arise from canonical protein-coding regions, cryptic antigens can originate from noncanonical ORFs, including upstream ORFs (uORFs), out-of-frame ORFs, alternative ORFs, and those within long non-coding RNAs (lncRNAs).

In this study, the authors analyzed a repertoire of 159 HLA class I-restricted MiHAs, comprising 130 ORFs, with approximately 27.7% classified as cryptic. Using Ribo-seq data from six Epstein-Barr virus-transformed B lymphoblastoid cell lines (EBV-LCLs), they assessed the sensitivity of Ribo-seq in detecting translation initiation sites (TISs) across these ORFs. The findings revealed that Ribo-seq effectively identified TISs in canonical ORFs and certain cryptic ORFs, particularly uORFs and alternative ORFs. However, it showed limited sensitivity for detecting out-of-frame ORFs and those within lncRNAs.

These results highlight that while Ribo-seq is a valuable tool for uncovering many translated regions, it has limitations in capturing the full spectrum of cryptic antigen sources. This underscores the need for complementary approaches to comprehensively identify potential T-cell antigens, which is crucial for advancing immunotherapeutic strategies.

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

Identification of Small Open Reading Frame Encoded Proteins from the Human Genome

Genomics, Proteomics & Bioinformatics, 2025

Kore H., Okano S., Datta K.K., Thorp J., Periasamy P., Divate M., Liyanage U., Hartel G., Nagaraj S.H., Gowda H.

This paper presents an integrated proteogenomics approach to systematically identify and validate small open reading frame (sORF)-encoded proteins (SEPs) in humans. While traditional annotations recognize approximately 20,500 protein-coding genes, recent studies suggest that numerous small ORFs, often located in untranslated regions (UTRs) of mRNAs and non-coding RNAs, may also be translated into functional proteins.

In this study, the authors analyzed publicly available ribosome profiling (Ribo-seq) and global proteomics datasets to establish protein-coding evidence for these sORFs. They predicted translation from 4,008 ORFs based on recurrent ribosome occupancy signals across multiple samples. Additionally, they identified 825 SEPs through proteomics data. Notably, some of these novel protein-coding regions were located in genome-wide association study (GWAS) loci associated with various traits and disease phenotypes. Furthermore, peptides from SEPs were found to be presented by major histocompatibility complex class I (MHC-I) complexes, similar to canonical proteins.

These findings expand the current catalog of protein-coding genes and suggest that SEPs may play significant roles in human biology and disease. The study underscores the importance of integrating Ribo-seq and proteomics data to uncover previously unannotated proteins, providing a valuable resource for future research into the functional roles of SEPs in cellular processes and disease mechanisms.

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

CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation and inflammatory signaling

PNAS, 2024

Halasz H., Malekos E., Covarrubias S., Yitiz S., Montano C., Sudek L., Katzman S., Liu S.J., Horlbeck M.A., Namvar L., Weissman J.S., Carpenter S.

In this paper, the authors explore the role of long noncoding RNAs (lncRNAs) in immune cell function. Using CRISPR interference (CRISPRi) screens in human monocytes, the researchers identified lncRNAs that influence both macrophage differentiation and the TLR4-NFκB inflammatory pathway.

A standout finding was the lncRNA LOUP, located upstream of the SPI1 gene, a key transcription factor in myeloid lineage commitment. LOUP was found to be a cis-regulator of SPI1 expression, affecting monocyte-to-macrophage differentiation. Additionally, LOUP contains small open reading frames (sORFs) that encode peptides capable of modulating the TLR4-NFκB signaling cascade, thereby influencing inflammatory responses.

This dual functionality of LOUP—as both a regulator of gene expression and a source of bioactive peptides—highlights the complex roles lncRNAs can play in immune regulation. The study underscores the utility of CRISPRi screens in uncovering multifunctional genomic elements, providing insights that could inform therapeutic strategies targeting immune-related diseases.

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

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