Micropeptides (also referred to as microproteins) are polypeptides with a length of less than 100-150 amino acids that are encoded by short open reading frames (sORFs). In this respect, they differ from many other active small polypeptides, which are produced through the posttranslational cleavage of larger polypeptides. In terms of size, micropeptides are considerably shorter than "canonical" proteins, which have an average length of 330 and 449 amino acids in prokaryotes and eukaryotes, respectively. Micropeptides are sometimes named according to their genomic location. For example, the translated product of an upstream open reading frame (uORF) might be called a uORF-encoded peptide (uPEP). Micropeptides lack an N-terminal signaling sequences, suggesting that they are likely to be localized to the cytoplasm. However, some micropeptides have been found in other cell compartments, as indicated by the existence of transmembrane micropeptides. They are found in both prokaryotes and eukaryotes. The sORFs from which micropeptides are translated can be encoded in 5' UTRs, small genes, or polycistronic mRNAs. Some micropeptide-coding genes were originally mis-annotated as long non-coding RNAs (lncRNAs). Given their small size, sORFs were originally overlooked. However, hundreds of thousands of putative micropeptides have been identified through various techniques in a multitude of organisms. Only a small fraction of these with coding potential have had their expression and function confirmed. Those that have been functionally characterized, in general, have roles in cell signaling, organogenesis, and cellular physiology. As more micropeptides are discovered so are more of their functions. One regulatory function is that of peptoswitches, which inhibit expression of downstream coding sequences by stalling ribosomes, through their direct or indirect activation by small molecules.
Identification Various experimental techniques exist for identifying potential sORFs and their translational products. These techniques are only useful for identification of sORF that may produce micropeptides and not for direct functional characterization.
RNA sequencing One method for finding potential sORFs, and therefore micropeptides, is through RNA sequencing (RNA-Seq). RNA-Seq uses next-generation sequencing (NGS) to determine which RNAs are expressed in a given cell, tissue, or organism at a specific point in time. This collection of data, known as a transcriptome, can then be used as a resource for finding potential sORFs. Because of the strong likelihood of sORFs less than 100 aa occurring by chance, further study is necessary to determine the validity of data obtained using this method.
Ribosome profiling (Ribo-Seq)
Ribosome profiling has been used to identify potential micropeptides in a growing number of organisms, including fruit flies, zebrafish, mice and humans. One method uses compounds such as harringtonine, puromycin or lactimidomycin to stop ribosomes at translation initiation sites. This indicates where active translation is taking place. Translation elongation inhibitors, such as emetine or cycloheximide, may also be used to obtain ribosome footprints which are more likely to result in a translated ORF. If a ribosome is bound at or near a sORF, it putatively encodes a micropeptide.
Mass spectrometry Mass spectrometry (MS) is the gold standard for identifying and sequencing proteins. Using this technique, investigators are able to determine if polypeptides are, in fact, translated from a sORF.
Proteogenomic applications Proteogenomics combines proteomics, genomics, and transciptomics. This is important when looking for potential micropeptides. One method of using proteogenomics entails using RNA-Seq data to create a custom database of all possible polypeptides. Liquid chromatography followed by tandem MS (LC-MS/MS) is performed to provide sequence information for translation products. Comparison of the transcriptomic and proteomics data can be used to confirm the presence of micropeptides.
Phylogenetic conservation Phylogenetic conservation can be a useful tool, particularly when sifting through a large database of sORFs. The likelihood of a sORF resulting in a functional micropeptide is more likely if it is conserved across numerous species. However, this will not work for all sORFs. For example, those that are encoded by lncRNAs are less likely to be conserved given lncRNAs themselves do not have high sequence conservation. Further experimentation will be necessary to determine if a functional micropeptide is in fact produced.
Validating protein-coding potential
Antibodies Custom antibodies targeted to the micropeptide of interest can be useful for quantifying expression or determining intracellular localization. As is the case with most proteins, low expression may make detection difficult. The small size of the micropeptide can also lead to difficulties in designing an epitope from which to target the antibody.
Tagging with CRISPR-Cas9 Genome editing can be used to add FLAG/MYC or other small peptide tags to an endogenous sORF, thus creating fusion proteins. In most cases, this method is beneficial in that it can be performed more quickly than developing a custom antibody. It is also useful for micropeptides for which no epitope can be targeted.
In vitro translation This process entails cloning the full-length micropeptide cDNA into a plasmid containing a T7 or SP6 promoter. This method utilizes a cell-free protein-synthesizing system in the presence of 35S-methionine to produce the peptide of interest. The products can then be analyzed by gel electrophoresis and the 35S-labeled peptide is visualized using autoradiography.
Databases and repositories There are several repositories and databases that have been created for both sORFs and micropeptides. A repository for of small ORFs discovered by ribosome profiling can be found at sORFs.org. A repository of putative sORF-encoded peptides in Arabidopsis thaliana can be found at ARA-PEPs. A database of small proteins, especially encoded by non-coding RNAs can be found at SmProt.
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