Identification and Validation of SmORF-Encoded Peptides by Genomics and Proteomics in Five Cyanobacteria

Small open reading frames (smORFs) and smORF-encoded peptides (SEPs) are now perceived as essential components alongside canonical genes and proteins. In cyanobacteria, SEPs were initially associated with photosynthesis and were later found to regulate metabolism and stress responses. However, SEPs have not been comprehensively identified in model cyanobacteria. Here, we used genomic and proteomic methods to identify and validate SEPs in five cyanobacteria. We integrated known SEPs from databases with genome-based predictions to construct a comprehensive data set. Generally, SEPs shared characteristics with canonical proteins in terms of amino acid frequency and codon bias. They also exhibited evolutionary features including paralogs within species and conservation across species, similar to canonical proteins. We found that most SEPs were related to photosynthesis, ribosomal proteins, and prokaryotic defense systems. Through SEP enrichment, we identified 817 SEPs with no previous evidence, and 103 novel SEPs through LC-MS/MS. They were identified as components of type II TA systems in cyanobacteria. The conserved SEP BolA might interact with Grx4 to mediate iron homeostasis. We also found several SEPs with potential functions, including sulfur transfer and protein translocation. In sum, our study excavates SEPs and suggests their biological roles in cyanobacteria.