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Updated: May 5, 2026
Identification of Circular RNAs using RNA Sequencing
Published on: November 14, 2019
Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides
Humberto J Ferreira1,2,3, Brian J Stevenson1,3,4, HuiSong Pak1,2,3
1Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland.
Abstract:
Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.
Insights
This study introduces a new method to find tumor-specific antigens from circular RNAs (circRNAs). These novel antigens could potentially boost anti-tumor T cell responses in cancer immunotherapy.
Area of Science:
- Immunology
- Genomics
- Cancer Research
Background:
- Circular RNAs (circRNAs) are non-coding RNAs with a unique structure.
- Some circRNAs can be translated into proteins, suggesting potential roles in disease.
- Aberrantly expressed circRNAs in cancer may represent novel sources of tumor-specific antigens.
Purpose of the Study:
- To develop and validate an immunopeptidomics workflow for identifying circRNA-derived peptides.
- To create a circRNA-specific protein reference database for enhanced analysis.
- To explore circRNAs as a source of tumor antigens for cancer immunotherapy.
Main Methods:
- Development of an immunopeptidomics workflow.
- Generation of a circRNA-specific protein FASTA reference.
- Application of stringent criteria including dual mass spectrometry engine identification and group-specific FDR.
- Validation of back-splice junction (BSJ)-spanning peptides using targeted MS and Sanger sequencing.
Main Results:
- Identification of 54 unique BSJ-spanning circRNA-derived peptides.
- Detection of these peptides in melanoma and lung cancer immunopeptidomes.
- Demonstration of a streamlined workflow for circRNA peptide identification.
Conclusions:
- The developed workflow effectively identifies circRNA-derived peptides.
- CircRNAs represent a promising, yet largely unexplored, source of tumor antigens.
- This work expands the repertoire of potential targets for cancer immunotherapy.
