Key highlights

• Micropeptides are small (less than 100 amino acid) proteins.
• Micropeptides are produced by translation of small open reading frames across the transcriptome.
• These proteins play significant roles in health and diseases including cancer and metabolic disorders.
• Ribosome profiling can be used to identify open reading frames with active ribosome translation.

Introduction

Over 20 years ago the Human Genome Project estimated that there were 20,000 to 25,000 protein coding genes¹, and approximately 18,000 have been verified by the Human Proteome Project². The identification of these proteins has historically relied on in silico screening, with set rules such as requiring a minimum length of 100 amino acids, followed by verification with mass spectrometry^1,2. A growing body of evidence has revealed that there is a second layer to the proteome: micropeptides that are shorter than 100 amino acids¹. Although previously assumed to be nonfunctional or artifacts, micropeptides have now been implicated in several disease processes. Their regulation has become an active area of drug development, with a growing number of companies aiming to alter their production or activity³.

In this eBlog we review the biology of micropeptides, their therapeutic potential, and state-of-the art methods for their detection.

Biology of micropeptides

As reviewed in one of our earlier eBlogs, peptides are produced from specific regions of the genome called open reading frames (ORFs). Historically, research has focused on long ORFs that encode for annotated proteins (also called a coding sequence or CDS). In addition to these long coding sequences there are millions of small ORFs (sORFs or smORFs) present across the transcriptome. They are often found in long non-coding RNAs, regulatory UTRs, introns, or in alternative reading frames of protein coding genes¹.

Just because a sORF is present on an RNA doesn’t necessarily mean that it associates with ribosomes and creates proteins. However, a subset of these can be productive, leading to the generation of micropeptides (also called sORF-encoded peptides or SEPs)⁴. These proteins typically lack multidomain structures and are hypothesized to mainly act within the cytoplasm^4,5.

The role of micropeptides in health

Although the role of micropeptides is still an active area of research, the functions of many have been identified across a wide range of biological processes, including:

• Hormone signaling: Elabela is secreted by stem cells and plays a role in cell differentiation¹.
• Regulation of canonical proteins: ASPRS is a small peptide that inhibits STAT3 activity by masking a phosphorylation site¹.
• Formation of protein complexes: ASDURF is a micropeptide that is required for the assembly of a chaperone protein complex¹.
• Molecular mimicry: STORM shares high sequence similarity with the binding region of SRP19 and may compete for binding to SRP19 targets¹. A similar phenomenon has been frequently observed in plants as well⁴.
• Immune system signaling: P155 is expressed in inflamed antigen-presenting cells and plays a role in T-cell priming¹.
• Membrane proteins: DWORF and PLN are two small proteins that localize to the sarcoplasmic reticulum and modulate calcium transport activity¹.

The role of micropeptides in disease

With their widespread production and roles in diverse cellular processes, micropeptides have been implicated in a number of diseases, including:

• Cancer: multiple micropeptides have been implicated in cancer progression, including SMIM30, which promotes metastasis, and ASAP, which promotes cancer cell proliferation⁷.
• Metabolic disorders: MOTS-c, a 16-amino-acid peptide, is linked to insulin resistance and diabetes, while SLN influences fat metabolism by regulating sarcoplasmic-reticulum calcium pumps⁸.
• Neurodegeneration: Humanin, one of the first micropeptides discovered, can limit apoptosis which provides benefits for Alzheimer’s disease⁹.

Due to these disease associations, there is growing interest in using micropeptides as diagnostic markers or directly as therapeutics^3,7. However, a major challenge with their widespread use in diagnosis and treatment is the detection of what micropeptides are produced in a given cell type or disease state.

Ribosome profiling is a micropeptide discovery engine

Ribosome profiling (Ribo-Seq) is a next-generation sequencing-based assay for measuring ribosome dynamics. This technique is based on ribosome footprinting, where RNAs are treated with a nuclease leading to the retention of only RNAs that are actively within the ribosome complex at the time of experiment¹⁰. After sequencing, bioinformatics analyses are performed to map where these ribosome protected footprints originate. Active sORFs with the potential to produce micropeptides can be identified from these data by looking for regions with 3-nucleotide periodicity (arising from translocation of ribosomes from codon to codon) and uniform read coverage along the length of the ORF, with increases in read density at the start and stop codons. As part of our ribosome profiling service, we provide the identification of active ORFs as a standard part of each project to support our partners in the identification of therapeutically-relevant micropeptides.

A major benefit of ribosome profiling for micropeptide identification is that it does not require any prior knowledge before the experiment is performed. This allows researchers and drug developers to perform unbiased screens to identify therapeutically relevant peptides and detect condition-specific changes in their translation.

Although ribosome profiling allows for the identification of physiologically relevant translation of sORFS, an orthogonal method like mass spectrometry is typically used for verification¹. Although mass spectrometry can confirm that the protein is present, standard approaches are not optimized for the detection of small peptides. This requires that specific protocol adaptations are done, such as non-enzyme based lysis or size enrichment on a gel, before performing the mass spectrometry¹¹.

Conclusion

Micropeptides expand our view of the proteome from a landscape dominated by large, well-annotated proteins to a complex architecture where tiny proteins play significant roles in physiology and disease. Their discovery challenges long-standing assumptions about gene annotation and is forming the basis for a new wave of therapeutic development. As assays like our optimized eRibo Pro continue to be used to profile different tissues and cell types, powerful AI models can be trained to help rapidly grow the micropeptide field to support biomarker identification and drug design.  If you would like to learn more about how our team can support your research into micropeptide biology, contact us today.

References

1. 1. Wright et al.
2. 2. Omenn et al.
3. 3. Zhong et al.
4. 4. Couso and Patraquim
5. 5. Crappé et al.
6. 6. Xiao et al.
7. 7. Zhou et al.
8. 8. Lu et al.
9. 9. Hassel et al.
10. 10. Brar and Weissman
11. 11. Kute et al.