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

Abstract

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.

RNA stabilization in hyperthermophilic archaea

“…2ʹ‐ O ‐methylation impairs phosphodiester bond cleavage resulting from the nucleophilic attack of a deprotonated 2ʹ OH group. Archaeal 2ʹ‐ O ‐methylations are usually introduced via C/D box sRNAs, or to a lesser degree, H/ACA box sRNAs, and elevated levels of these guide RNAs have been observed for hyperthermophiles . Individual C/D box sRNAs are characterized by two pairs of conserved C box (C and Cʹ: 5ʹ‐RUGAUGA‐3ʹ) and D box (D and Dʹ: 5ʹ‐CUGA‐3ʹ) sequences that flank unstructured guide sequences.…”

“…Recently, Lui et al . have used gene prediction models and RNA‐seq data to identify 526 C/D box sRNA genes in six Pyrobaculum species . The evolutionary history of guide sequences provides a fascinating view of the changing methylation potential in these hyperthermophilic organisms.…”

“…Dual guide interactions are proposed to aid folding and stabilization of nascent rRNAs, and subsequent 2ʹ‐ O ‐methylation of the rRNA duplex region greatly enhances its melting temperature . In addition to rRNAs, other C/D box sRNA targets are tRNAs with 2ʹ‐ O ‐methylation occurring at structurally conserved positions . C/D box sRNA sequences have also been found in introns of tRNA molecules and were shown to be able to direct modification of tRNA anticodon positions in cis .…”

“…Archaeal 2ʹ-O-methylations are usually introduced via C/D box sRNAs, or to a lesser degree, H/ACA box sRNAs, and elevated levels of these guide RNAs have been observed for hyperthermophiles. 29,30 Individual C/D box sRNAs are characterized by two pairs of conserved C box (C and Cʹ: 5ʹ-RUGAUGA-3ʹ) and D box (D and Dʹ: 5ʹ-CUGA-3ʹ) sequences that flank unstructured guide sequences. The sRNAs guide a ribonucleoprotein (RNP) complex consisting of L7Ae, Nop56/58 (also called Nop5), and fibrillarin subunits to their RNA targets, and the methyltransferase fibrillarin introduces the 2ʹ-O-methylation modification 5 nt upstream of the first complementary base of the D or Dʹ box sequence.…”

“…Recently, Lui et al have used gene prediction models and RNA-seq data to identify 526 C/D box sRNA genes in six Pyrobaculum species. 29 The evolutionary history of guide sequences provides a fascinating view of the changing methylation potential in these hyperthermophilic organisms. As every C/D box sRNA features two guide sequences, one upstream of the D box and one upstream of the Dʹ box, their coordinated guidance of methylation targets was proposed.…”