FScanR reveals widespread programmed ribosomal frameshifting consistent with neutral evolution in a ciliate model.

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism that allows multiple functionally distinct proteins to be synthesized from a single mRNA transcript. Although PRF is widespread among both prokaryotic and eukaryotic organisms, accurately assessing its full scope in biological systems using conventional genomic or proteomic data alone remains challenging. To address this, we have developed FScanR, a computational tool that identifies PRF events with high confidence by systematically comparing in-frame and out-of-frame nucleotide sequences with corresponding peptide datasets. Our results show that (1) FScanR detects PRF events in protein-coding genes across phylogenetically distinct organisms, enabling us to characterize the sequence features surrounding the PRF sites; (2) euplotid ciliates exhibit widespread PRF events, with no significant bias in GC content or inter-site distance observed in genomic regions flanking the PRF sites; (3) genes associated with PRF events participate in critical physiological processes, including phosphorylation, ubiquitination and metabolic regulation; and (4) PRF in euplotids occurs across genes with varying expression levels and appears to be under neutral selection. These findings highlight the value of FScanR in revealing recoding mechanisms and offer new insights into the evolutionary dynamics of PRF in single-celled eukaryotes.