Methylation guide RNA evolution in archaea: structure, function, and genomic organization of 110 C/D box sRNA families across six Pyrobaculum species

Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2′- O -methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum , particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches, and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.