For decades, the central dogma of molecular biology established a neat separation: messenger RNAs carried coding information for proteins, while non-coding RNAs acted as regulators. Long non-coding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides lacking obvious coding potential, were considered archetypal regulatory RNAs. However, advances in next-generation sequencing (NGS), ribosome profiling, and functional proteomics are dismantling this dichotomy. Increasingly, lncRNAs are found to encode short, biologically active proteins (micropeptides) that play roles in cellular physiology and disease. This discovery is forcing a redefinition of what “coding” and “non-coding” mean, and it is expanding the boundaries of transcriptomics and proteomics simultaneously1.
Several human lncRNAs have been shown to encode short peptides (often 10–100 aa) with measurable biological activity in cells and in vivo. In the validated cases, translation evidence from ribosome profiling or ribosome–nascent chain complex sequencing is typically paired with direct peptide detection by tandem mass spectrometry and with functional assays (e.g., start-codon disruption or ORF-specific frameshift) to establish peptide-specific phenotypes1.
Micropeptides are frequently low in abundance and often hydrophobic, with a strong tendency to associate with membranes such as the sarcoplasmic reticulum, endoplasmic reticulum, or mitochondrial inner membranes. This localization helps explain their prominent roles in organelle-proximal signalling, ranging from the regulation of mitochondrial respiration and fission to calcium handling in muscle and heart, but it also complicates their study. Hydrophobicity and small size limit epitope availability, making antibody generation difficult, and they reduce recovery in mass spectrometry workflows, which collectively depresses routine detectability2-4.
Their translation often departs from canonical models. Instead of relying solely on cap-dependent initiation, many of these short ORFs exploit internal ribosome entry sites or m6A-driven initiation, allowing them to be selectively translated under stress or specific signaling conditions. This adds a layer of context dependency to their biology, helping explain why these peptides can emerge prominently in some conditions such as cancer, immunity, or stress responses5,6.
Finally, an added complexity is that lncRNA loci can be bi-functional: the RNA transcript may exert regulatory effects through RNA–protein or RNA–RNA interactions, while simultaneously encoding a peptide with distinct activity. Disentangling these layers requires targeted strategies such as start-codon mutagenesis or ORF-specific CRISPR editing to confirm that observed phenotypes truly derive from the peptide rather than from the RNA7,8.
Functional snapshots
Cancer. Several lncRNA-encoded peptides influence tumorigenesis. In colorectal cancer, HOXB-AS3 encodes a 53–amino acid peptide that binds hnRNPA1 to antagonize PKM splicing and suppress tumor growth7. By contrast, LINC00467 produces ASAP (94 aa), which interacts with ATP synthase subunits to boost oxidative metabolism and drive proliferation9. In breast cancer, LINC00908 encodes ASRPS (60 aa), which blocks STAT3 phosphorylation and angiogenesis10. An additional example includes UBAP1-AST6, whose knockout reduces lung-cancer growth8. Collectively, these peptides illustrate that lncRNAs can act as both tumor suppressors and oncogenes through peptide translation.
Immunity and inflammation. lncRNA-derived micropeptides also modulate immune responses. MIR155HG encodes miPEP155 (17 aa), which binds HSC70 and disrupts the HSC70–HSP90 machinery, altering antigen presentation and T-cell priming 11. This example highlights how small peptides hidden within lncRNAs can regulate professional antigen-presenting cells and immune tolerance.
Cardiovascular and muscle biology. Muscle-specific lncRNAs have been shown to encode micropeptides that fine-tune calcium handling. Myoregulin, a 46–amino acid peptide, inhibits the SERCA Ca²⁺ pump, while DWORF enhances its activity. Both reshape calcium cycling in muscle and cardiac cells, directly linking hidden coding potential to performance and stress responses2,3.
There are several databases listing lncRNA-encoded micropeptides. Some are lncRNA-specific, others are broader sORF/alt-protein databases that include lncRNA-derived peptides. We will mention two in this blog post:
- LncPep, a dedicated database for lncRNA-encoded peptides, integrating evidence from CPAT/CPC2, m6A, Ribo-seq and TIS annotations across 39 species12.
- ncEP, a manually curated collection of experimentally validated peptides/proteins encoded by non-coding RNAs (includes human lncRNAs), with links to original papers13.
NGS methods for identifying lncRNA-encoded micropeptides Total RNA-seq workflows such as the NEXTFLEX™ Small RNA-seq kit v4, sequences ribosome-protected fragments to pinpoint actively translated ORFs with nucleotide resolution and triplet periodicity.
Together, these NGS methods directly support translation calls, and they are complemented by analysis pipelines such as RiboTaper, ORF-RATER, and metrics like FLOSS to assess coding status. Orthogonal validation comes from targeted or discovery mass spectrometry and epitope tagging at endogenous loci. This workflow has become standard for proving the existence of lncRNA-encoded micropeptides1.
On the prediction side, widely used computational tools (CPAT, CPC/CPC2, PhyloCSF, COME, ORF Finder) help triage candidates prior to experimental work.
Conclusion
The evidence summarized here shows that some transcripts annotated as non-coding do encode functional micropeptides, and the coding/non-coding divide is far more permeable than once assumed. Standard RNA-seq remains essential for charting lncRNA transcription, but determining which lncRNAs produce peptides, and whether those peptides matter, requires NGS methods that measure the process of mRNA translation into proteins (Ribosome profiling and others), proteomics to confirm peptide identity, and ORF-targeted genetics to attribute function to the peptide rather than the RNA.
From a practical standpoint, analysis pipelines and annotations should stop discarding lncRNA sORFs by default, integrate ribosome-profiling evidence when interpreting differential expression, and plan validation with methods suited to low-abundance, membrane-associated peptides. Adopting these approaches reframes ‘non-coding’ as a provisional annotation subject to experimental verification, enabling systematic discovery and confirmation of lncRNA-encoded micropeptides.
References
- Xiao, Y., et al. (2024). Long non-coding RNA-encoded micropeptides: functions, mechanisms and implications. Cell Death Discov 10, 450. doi:10.1038/s41420-024-02175-0.
- Anderson, D.M., et al (2015). A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell.160(4):595-606. doi:10.1016/j.cell.2015.01.009.
- Nelson, B.R., et al. (2016) A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science. 351,271-275. doi:10.1126/science.aad4076.
- Slavoff, S., et al. (2013). Peptidomic discovery of short open reading frame–encoded peptides in human cells. Nat Chem Biol 9, 59–64 (2013). doi:10.1038/nchembio.1120.
- Charpentier, M., et al (2016). IRES-dependent translation of the long non coding RNA meloe in melanoma cells produces the most immunogenic MELOE antigens. Oncotarget.7(37):59704-59713. doi: 10.18632/oncotarget.10923.
- Wu, S., et al (2020). A Novel Micropeptide Encoded by Y-Linked LINC00278 Links Cigarette Smoking and AR Signaling in Male Esophageal Squamous Cell Carcinoma. Cancer Res. 80:2790–803. doi: 10.1158/0008-5472.CAN-19-3440.
- Huang J.Z., et al. (2017).A peptide encoded by a putative lncRNA HOXB-AS3 suppresses colon cancer growth. Mol Cell. 2017. 68(1)171-184. doi:10.1016/j.molcel.2017.09.015.
- Lu, S., et al (2019). A hidden human proteome encoded by 'non-coding' genes. Nucleic Acids Res. 47(15):8111-8125. doi: 10.1093/nar/gkz646.
- Ge, Q., et al. (2021). Micropeptide ASAP encoded by LINC00467 promotes colorectal cancer progression by directly modulating ATP synthase activity. J Clin Invest. 131(22):e152911. doi:10.1172/JCI152911.
- Wang, Y., et al. (2020). LncRNA-encoded polypeptide ASRPS inhibits triple-negative breast cancer angiogenesis. J Exp Med. 217 (3): e20190950. doi: 10.1084/jem.20190950.
- Niu, L., et al. (2020). A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation. Sci Adv. 6,eaaz2059, doi: 10.1126/sciadv.aaz2059.
- Liu, T., et al (2022). LncPep: A Resource of Translational Evidences for lncRNAs. Front Cell Dev Biol. 10:795084. doi: 10.3389/fcell.2022.795084.
- Liu, H., et al (2020). ncEP: A Manually Curated Database for Experimentally Validated ncRNA-encoded Proteins or Peptides. J Mol Biol. 432(11):3364-3368. doi: 10.1016/j.jmb.2020.02.022.
