C/D box sRNA-guided 2'-O-methylation patterns of archaeal rRNA molecules

Abstract

Background: In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.

Results: We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.

Conclusions: This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.

Fig. 1

Distribution of 2′-O-ribose methylation sites in archaeal 23S rRNA. Predicted sites of sRNA directed 2′-O-ribose methylation (Additional Files 1 and 3) from seven archaeal species (listed in Table 1) were mapped onto the consensus secondary structure of the archaeal 23S rRNA [21]. The multiple occurrences of methylation at a given nucleotide position is indicated by increasing dot size (i.e. methylation targets found in one to six organisms). The color of the dots (from blue to red) indicates increasing methylation frequency within a nine nucleotide window. The archaeal nucleotide alignment positions of sites of modification discussed in the context of 23S rRNA structure-function are indicated

Fig. 2

Distribution of 2′-O-ribose methylation sites in archaeal 16S rRNA. Predicted sites of sRNA directed 2′-O-ribose methylation (Additional Files 1 and 4) from seven archaeal species listed in Table 1 were mapped onto the consensus secondary structure of the archaeal 16S rRNA [21]. The multiple occurrences of methylation at a given nucleotide position is indicated by increasing dot size (i.e. methylation targets found in one to six organisms). The color of the dots (from blue to red) indicates increasing methylation frequency within a nine nucleotide window. The archaeal nucleotide alignment positions of sites of modification discussed in the context of 16S rRNA structure-function are indicated

Fig. 3

Complexes between sRNA guides and targets at sites of conserved 2′-O-ribose methylation in rRNA. a Conserved methyl modification of position 23S rRNA C/U2880. The complementarities between the D' and D guide regions of Pca sR30, Tte sR152, Sac sR13, Mka sR3, Neq sR10 and Iho sR6 with the 23S rRNA region around position C/U2880 (Eco position C2606; see Additional file 3) are illustrated with position 2880 aligned in the middle and labelled in red. The sRNA sequences for each species are listed below the rRNA sequences with the C and C' boxes outlined with blue and the D' and D box sequences outlined with green. The predicted positions of methyl modification are indicated by “Me” in red within the rRNA sequences. The D' guide of Tte sR112 has no significant complementarity to rRNA (empty guide). The D' guide-target complementarity of Sac sR13 exhibits a mismatch basepair at the predicted site of modification (C2847; MM:mismatch) and is likely not modified. b Conserved methyl modifications at positions 16S A512 and G491. The complementarities between the D' and D guide regions of Tte sR114, Iho sR21, Pca sR28 and Sac sR115 to the regions around 16S A512 (left of center) and G491 (right of center) are illustrated. These modification sites correspond to positions A535 and G515 in helix 18 of Eco 16S rRNA (Additional file 3). The sRNA C and C', and D' and D boxes are outlined with blue and green respectively. The predicted sites of methyl modification are indicated by (Me) in red. The sequence complementarity between the D guide of Iho sR21 and the region of around 16S G491 is disrupted by three GU base pairs (0) which likely compromises methylation activity; this Iho sR21 site was not considered to be a robust prediction in our analysis

Fig. 4

Sequence conservation and divergence within orthologous sRNA genes. The notation for box sequences and predicted positions of rRNA modification are described in the legend to Fig. 3. a The Mka sR39 and sR119 sRNAs are derived from duplicate genes and exhibit a high degree of end-to-end sequence similarity. The sR88 has a D box guide that exhibits high similarity to the D' guides of sR39/119 and a D' guide that exhibits only residual similarity to the D guide of sR39/119. The predicted D and D' target sites for sR88 are 23S G3118 and C3169; in our analysis, 16S G152 was not considered a D guide-target for sR88 because it did not meet the prediction criteria. b The Mka sR24 and 25 sequences are derived from duplicated genes. The 5' and 3' regions of the D guides are affected by indels that shift the site of modification between C179 and G176. c The Mka D' guide of sR56 and the D guide of sR103 are highly similar in sequence and are predicted to guide modification to 23S G2759. The D guide of sR56 and the D′ guide of sR103 are unrelated and predicted to guide modification to positions 23S A2849 and G2840 respectively

Fig. 5

A chaperone function for C/D box sRNAs in long distant 16S rRNA folding. In Nanoarchaeum equitans, the guide regions of sR17 and sR15 exhibit extended complementary to the two strands of helix 28 and 30 of 16S rRNA respectively. The sRNA-rRNA interactions (left) position the distant rRNA sequences into close proximity. Release of the sRNAs following methylation at positions A915, G930 G1218 and U1371 is postulated to play a critical role in the efficient folding of helices 28, 29 and 30 in the nascent 16S rRNA (right)