Control of box C/D snoRNP assembly by N ⁶ ‐methylation of adenine

Abstract

N6‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N6‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N6‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N6mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.

Sequence determinants of the folding properties of box C/D kink-turns in RNA

“…We have observed that each k-turn whose sequence prevents ion-dependent folding will nevertheless fold in response to the binding of L7Ae, including the Af box C/D k-turn. Similarly, we have previously shown that human box C/D k-turns fold on binding the mammalian L7Ae homolog 15.5k protein (Huang et al 2017).…”

“…U:U and 4b:4n = G:C are strongly conserved (Huang et al 2017). In the assembly of the box C/D snoRNP the first event is the binding of L7Ae (15.5k in humans), thereby generating the kinked conformation of the k-turn.…”

“…Thus assembly of the box C/ D snoRNP can in principle be controlled at the stage of the binding of L7Ae/15.5k to the k-turn. We have recently shown that in a subset of human box C/D sites this can be regulated by N 6 -methylation of A1n, which disrupts the G•A trans sugar-Hoogsteen base pair required to form the k-turn (Huang et al 2017). However, methylation of the A1n requires that the nucleotide at −1n be cytosine, to create a GAC target for the METTL3-METTL14 methyl transferase (Dominissini et al 2012;Meyer et al 2012) and thus for these box C/D species −1b:−1n = G:C.…”

N6‐Methyladenosine‐Sensitive RNA‐Cleaving Deoxyribozymes

“…To explore the response of these m 6 A‐sensitive DNA enzymes on longer RNA substrates, we chose a family of C/D box snoRNAs for which the influence of m 6 A on snoRNP assembly has been suggested . We investigated the DNA‐catalyzed cleavage of full‐length non‐coding RNAs, including human SNORD29, SNORD41, and SNORD44, addressing the adenosine of the key trans Hoogsteen‐sugar A:G base‐pair that is essential for folding of the k‐turn structure and binding of the 15.5k protein and that is reported to be N 6 ‐methylated . The VMA15 and VMC10 DNA enzymes were designed to cleave upstream of the putative m 6 A (shown in red in Figure a), and the cleavage of unmodified and methylated RNA was compared (Figure and Figure S11) .…”

N6‐Methyladenosine‐Sensitive RNA‐Cleaving Deoxyribozymes

“…To explore the response of these m 6 A-sensitive DNA enzymes on longer RNA substrates, we chose a family of C/D box snoRNAs for which the influence of m 6 A on snoRNP assembly has been suggested. [23] We investigated the DNA-catalyzed cleavage of full-length non-coding RNAs, including human SNORD29, SNORD41, and SNORD44, addressing the adenosine of the key trans Hoogsteen-sugar A:G base-pair that is essential for folding of the k-turn structure and binding of the 15.5k protein, and that is reported to be N 6 -methylated. [23] The VMA15 and VMC10 DNA enzymes were designed to cleave upstream of the putative m 6 A (shown in red in Figure 3a), and the cleavage of unmodified and methylated RNA was compared (Figure 3, S11).…”