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.