Circular-pre-rRNA-dependent and -independent rRNA maturation pathways in Archaea

Ribosome biogenesis in archaea is uniquely characterized by the formation of circular pre-rRNA intermediates, a process dependent on bulge-helix-bulge (bhb) motifs—structured RNA elements located in the processing stems flanking the rRNA and critical for cleavage and ligation by the tRNA splicing machinery. In this study, we confirm the existence of circular-dependent rRNA maturation pathways across diverse archaeal species. In addition, we also reveal the presence of circular-independent rRNA maturation pathways. Using a pan-archaeal assay, we detected circular pre-16S rRNA in most analysed species, but notably, Thermoplasma acidophilum lacks both circular pre-16S intermediate and a bhb motif in the 16S processing stem, indicating a novel, bhb- and circular-independent pre-16S maturation mechanism. In Haloarcula marismortui, we identified hybrid pathways, where different operons utilize either circular-dependent or -independent maturation routes. Additionally, we uncovered unprecedented diversity in bhb motifs, with central helix lengths ranging from 3 to 6 nucleotides, contrasting with the canonical 4-nucleotide helix typically observed in the context of tRNA splicing. These findings expand our understanding of rRNA maturation in archaea, demonstrating that circular pre-16S rRNA formation in archaea is not a universal requirement and highlighting the adaptability of archaeal ribosome biogenesis.