Abstract
ABSTRACT N6-methyladenine is the most common covalent modification in cellular RNA species, with demonstrated functional consequences. At the molecular level this methylation could alter local RNA structure, and/or modulate the binding of specific proteins. We have previously shown that trans-Hoogsteen-sugar (sheared) A:G base pairs can be completely disrupted by methylation, and that this occurs in a sub-set ofD/D k-turn structures. In this work we have investigated to what extent sequence context affects the severity with which inclusion of N6-methyladenine into different A:G base pairs of a standard k-turn affects RNA folding and L7Ae protein binding. We find that local sequence has a major influence, ranging from complete absence of folding and protein binding to a relatively mild effect. We have determined the crystal structure of one of these species both free and protein-bound, showing the environment of the methyl group and the way the modification is accommodated into the k-turn structure.
Highlights
N6-methyladenine (Figure 1(a)) is very widespread in cellular RNA [1,2,3]
We have previously shown that trans-Hoogsteen-sugar A:G base pairs can be completely disrupted by methylation, and that this occurs in a sub-set ofD/D k-turn structures
We have synthesized a series of variants of the standard k-turn Kt-7 from Haloarcula marismortui in which adenine has been substituted by N6-methyladenine
Summary
N6-methyladenine (Figure 1(a)) is very widespread in cellular RNA [1,2,3]. It is commonly found in eukaryotic mRNA, lncRNA species including Xist [2,3] and MALAT1 [4,5] as well as in viral RNA [6]. In principle the presence of N6-methyladenine in cellular RNA could be recognized in two alternative ways. A second possibility is that the addition of the methyl group might directly affect the structure of the RNA and so alter its ability to assemble into a functional species [14,15,16,17,18]. Sheared G:A base pairs are frequently found in key helical junctions in RNA structure, and most notably within the core of kink turns (k-turns) as tandem G:A, A:G base pairs [19,20]
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