The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer

Abstract

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)22 forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)9AGG (CGG)12AGG(CGG)97, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3' part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.

Figure 1

Analysis of CGG-RNA structure. S1 and T1 nuclease digestion of CGG-RNA. S1 and T1 treatment of RNA containing (CGG)₂₂ and (CGG)₉AGG(CGG)₁₂AGG(CGG)₉₇ [referred to as (CGG)₁₂₀]. The sizes of the S1 and T1 cleavage products were determined by comparison with both a pBR322 MspI digest and the background of T1 cleavage products resulting from a small amount of unstructured RNA in the reaction. T1B, S1B and DB refer to reactions carried out in the T1 nuclease, S1 nuclease and Dicer buffers as described in Materials and Methods.

Figure 2

Diagrammatic representation of the sequence of the (CGG)₉AGG(CGG)₁₂AGG(CGG)₉₇ RNA and the structures consistent with the S1 and T1 cleavage products. The repeat region is shown in black and the flanking sequence in gray. The arrows indicate the regions of nuclease cleavage. The tetrads and base pairs shown are for illustrative purposes only and are not meant to represent the actual number of hydrogen bonds formed.

Figure 3

Effect of CGG-RNA on PKR activation. (A) Phosphorylation of partially purified human PKR in vitro in response to added RNAs. The data are expressed as the amount of phosphorylated PKR relative to the endogenous levels of phosphorylated material present in the absence of added RNA. The level of PKR phosphorylation in response to added RNA for poly(I):poly(C) is indicated by triangles, (AUU)₈₈ by crosses, (CGG)₂₂ by diamonds and (CGG)₉AGG(CGG)₁₂AGG(CGG)₉₇ by squares. (B) The amount of phosphorylated eIF2α in cells transfected with in vitro synthesized RNA. The data are expressed as the amount of phosphorylated eIF2α at the indicated time points relative to the amount before addition of the RNA. The gray bars indicate the amount of phosphorylated eIF2α in cells transfected with poly(I):poly(C) and the black bars the amount detected in cells transfected with the same amount of (CGG)₉AGG(CGG)₁₂AG G(CGG)₉₇ RNA. (C) The amount of phosphorylated eIF2α in cells expressing an otherwise identical transcript containing either 0 (FRT0) or 176 (FRT176) CGG repeats. A small increase in the amount of phosphorylated eIF2α is seen in both cell lines at 48 h but, at least in the case of the FRT176 cell line, this does not seem to be related to expression of the CGG-containing RNA since more phosphorylated product is seen in the absence of dox than in its presence.

Figure 4

Dicer digestion of CGG-RNA. Bona fide single- and double-stranded RNAs were digested alongside (CGG)₉AGG(CGG)₁₂AGG(CGG)₉₇ RNA with human recombinant Dicer enzyme as described in Materials and Methods. The relative amount of ∼22 bp Dicer product produced was calculated by comparing the amount of this product with the amount of undigested material remaining.