Abstract
2'-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate-guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.
Highlights
In a wide variety of cellular processes, ranging from biosynthesis to signalling and regulation of gene expression, RNA is chemically modified both co- and post-transcriptionally
Using the Pyrococcus furiosus (Pf) sR26 guide RNA, whose corresponding sRNP methylates substrate D’ more efficiently than substrate D, we demonstrated that methylation levels depend on — among other factors — the nature of the first base-paired nucleotide of the substrate (Graziadei et al, 2016)
Using nuclear magnetic resonance (NMR), small angle X-ray (SAXS) and neutron (SANS) scattering data, we demonstrate that, in the context of the sRNP complex, the affinity of fibrillarin for the substrate depends on the RNA sequence beyond the methylation site
Summary
In a wide variety of cellular processes, ranging from biosynthesis to signalling and regulation of gene expression, RNA is chemically modified both co- and post-transcriptionally. Using nuclear magnetic resonance (NMR), small angle X-ray (SAXS) and neutron (SANS) scattering data, we demonstrate that, in the context of the sRNP complex, the affinity of fibrillarin for the substrate depends on the RNA sequence beyond the methylation site. This difference in affinity is explained by the energetics of a global conformational transition of the sRNP from an inactive to an active state and provides a further route, besides the modulation of product dissociation described previously (Graziadei et al, 2016), to tune RNA methylation levels. To derive these results we developed an ensemble structure-calculation method that exploits the ability of integrative structural biology in solution to reveal and characterize conformational equilibria
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