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Updated: Jun 3, 2025
An Integrated Approach for Microprotein Identification and Sequence Analysis
Published on: July 12, 2022
Pervasiveness of Microprotein Function Amongst Drosophila Small Open Reading Frames (SMORFS)
Ana Isabel Platero1, Jose Ignacio Pueyo2, Sarah Anne Bishop1,2
1Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, CSIC, 41013 Sevilla, Spain.
Abstract:
Small Open Reading Frames (smORFs) of less than 100 codons remain mostly uncharacterised. About a thousand smORFs per genome encode peptides and microproteins about 70-80 aa long, often containing recognisable protein structures and markers of translation, and these are referred to as short Coding Sequences (sCDSs). The characterisation of individual sCDSs has provided examples of smORFs' function and conservation, but we cannot infer the functionality of all other metazoan smORFs from these. sCDS function has been characterised at a genome-wide scale in yeast and bacteria, showing that hundreds can produce a phenotype, but attempts in metazoans have been less successful. Either most sCDSs are not functional, or classic experimental techniques do not work with smORFs due to their shortness. Here, we combine extensive proteomics with bioinformatics and genetics in order to detect and corroborate sCDS function in Drosophila. Our studies nearly double the number of sCDSs with detected peptides and microproteins and an experimentally corroborated function. Finally, we observe a correlation between proven sCDS protein function and bioinformatic markers such as conservation and GC content. Our results support that sCDSs peptides and microproteins act as membrane-related regulators of canonical proteins, regulators whose functions are best understood at the cellular level, and whose mutants produce little, if any, overt morphological phenotypes.
Insights
This study characterizes small Open Reading Frames (smORFs) in Drosophila, nearly doubling the number with known functions. These short Coding Sequences (sCDSs) encode microproteins regulating canonical proteins at the cellular level.
Area of Science:
- Genomics
- Proteomics
- Molecular Biology
Background:
- Small Open Reading Frames (smORFs), defined as <100 codons, are largely uncharacterized in metazoans.
- Short Coding Sequences (sCDSs) encode peptides/microproteins (70-80 aa) with potential structures and translation markers.
- Genome-wide functional studies in yeast/bacteria show sCDSs can impact phenotypes, but metazoan efforts are limited.
Purpose of the Study:
- To detect and corroborate the function of sCDSs in Drosophila using integrated experimental and computational approaches.
- To expand the repertoire of characterized smORFs and their encoded peptides/microproteins.
- To investigate the relationship between sCDS function and bioinformatic features.
Main Methods:
- Combined proteomics, bioinformatics, and genetics in Drosophila.
- Extensive proteomic analysis to detect peptides and microproteins from sCDSs.
- Bioinformatic analysis including conservation and GC content assessment.
Main Results:
- Nearly doubled the number of sCDSs with detected peptides/microproteins and experimentally corroborated function.
- Identified a correlation between proven sCDS protein function and bioinformatic markers (conservation, GC content).
- Observed that sCDS peptides/microproteins function as membrane-related regulators of canonical proteins.
Conclusions:
- sCDSs encode functional peptides and microproteins, often acting as regulators at the cellular level.
- The function of smORF-encoded proteins is often subtle, not leading to overt morphological phenotypes.
- Bioinformatic markers can predict functional sCDSs, aiding future characterization efforts.
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