Analysis and perturbation of C/D box sRNA-guided 2'-O-methylation patterns of Sulfolobus acidocaldarius ribosomal RNAs
General Genetics and Functional Genome Biology
Final Report Abstract
2′-O-methylation is the most abundant modification of ribosomal RNAs in hyperthermophilic Archaea. Archaea utilize ribonucleoprotein (RNP) complexes containing small (s)RNAs to identify targets for 2′-O-methylation. These RNAs are termed C/D box sno-like RNAs (C/D box sRNA) in archaea and resemble their eukaryotic snoRNA counterparts in structure and function. They are characterized by conserved sequence elements called box C and C′ and box D and D′. These elements fold into a ubiquitous RNA structural motif, the kink-turn (k-turn). Two k-turn elements flank short, highly variable guide sequences that exhibit complementarity to sequences within rRNA. Consequently, each C/D box sRNA contains a combination of two guide sequences that specify target methylation sites via base complementarity, directing the activity of the methyltransferase fibrillarin. Two copies of the L7Ae protein bind and stabilize the k-turn motifs. In this project, we aimed to investigate the targeting potential of the C/D box sRNAs that were identified in S. acidocaldarius. Additionally, we aimed to establish a methodology that allows for the global analysis of methylation sites. After submission of this proposal, several groups reported methods to achieve this goal and we were able to utilize available methodology in collaboration with Prof. Gordon Carmichael (University of Connecticut). However, these analyses also highlighted problems for the differentiation of modification sites and artefacts caused by highly stable rRNA structures. Analysis of the individual C/D box sRNA guide sequences of S. acidocaldarius allowed for two surprising observations. First, we discovered that C and D boxes are often formed due to overlap with start and stop codons of proteincoding genes allowing for accelerated evolution of the C/D box guide pairs. Second, individual k-turns, representing half C/D box sRNA molecules, were found to be fused to mRNAs, impacting their stability and allowing for their gene regulation. Regulated genes include l7ae and other C/D box sRNA binding partners, enabling negative feedback. These finding highlighted that the accelerated evolution of C/D box sRNA genes allow for a 2′-O-methylation landscape of rRNAs with high plasticity and the potential to evolve novel paired methylation sites that can stabilize rRNA interactions during folding.
Publications
- Plasticity of archaeal C/D box sRNA biogenesis. Mol Microbiol. 2017 Jan;103(1):151-164
Tripp V, Martin R, Orell A, Alkhnbashi OS, Backofen R, Randau L
(See online at https://doi.org/10.1111/mmi.13549) - RIP-Seq Suggests Translational Regulation by L7Ae in Archaea. mBio. 2017 Aug 1;8(4). pii: e00730-17
Daume M, Uhl M, Backofen R, Randau L
(See online at https://doi.org/10.1128/mBio.00730-17) - Noncoding RNAs in Archaea: Genome-Wide Identification and Functional Classification. Methods Enzymol. 2018;612:413-442
Buddeweg A, Daume M, Randau L, Schmitz RA
(See online at https://doi.org/10.1016/bs.mie.2018.08.003) - Unique Archaeal Small RNAs. Annu Rev Genet. 2018 Nov 23;52:465-487
Gomes-Filho JV, Daume M, Randau L
(See online at https://doi.org/10.1146/annurev-genet-120417-031300) - RNA stabilization in hyperthermophilic archaea. Ann N Y Acad Sci. 2019 Jul;1447(1):88-96
Gomes-Filho JV, Randau L
(See online at https://doi.org/10.1111/nyas.14060)
