Molecular phylogenetics, distribution and evolution of the TPP riboswitch

Thiamine pyrophosphate (TPP) riboswitches are the only riboswitch class identified in eukaryotes. They therefore provide a unique model for reconstructing the evolution of RNA-based gene regulation across life's domains. We investigated the molecular evolution, structural diversification, and genomic-context reassignment of the TPP riboswitch across archaea, bacteria, cyanobacteria, fungi, algae, and land plants using phylogenetic reconstruction, covariance-model-guided sequence analysis, comparative secondary-structure analysis, genomic-context annotation, and covariation-based inference. Our analyses establish an early prokaryotic origin of the TPP riboswitch as a 5'- UTR-associated regulatory element. Archaea retain the most compact ancestral scaffold. The transition to bacteria is characterized by extensive compensatory evolution. Bacteria preserve the canonical fold but exhibit the strongest covariation. Within bacteria, cyanobacteria exhibit a reduced architecture while retaining a conserved ligand-binding core, indicating an early reform event. Methylogaea oryzae branches within the cyanobacterial clade, identifying horizontal acquisition of a thiC-TPP riboswitch cassette. The transition into the green lineage is marked by the repositioning of the riboswitch from the ancestral 5'-UTR to the 3'-UTR. This architecture is found in the charophyte Closterium, indicating that the plant-type 3'-UTR state predates land plants. Fungi retain mainly 5'-UTR organization but also contain a distinct 3'-UTR NMT1/thi5-associated clade, defining a second eukaryotic regulatory configuration derived through horizontal transfer from early green lineages. These results suggest a continuous evolutionary trajectory of the TPP riboswitch from an ancestral prokaryotic 5'-UTR regulator to the green-lineage repositioning of the 3'-UTR, to fungal functional reassignment, and the secondary emergence of an alternative 3'-UTR state.