Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference

Abstract

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington’s disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart.

CAG repeat RNA as an auxiliary toxic agent in polyglutamine disorders

“…13,16 More recently, transcripts harboring translated CAG repeat expansions have been observed to form intranuclear MBNL1-positive RNA foci in human HD fibroblasts. 21 This result is in agreement with earlier reports describing nuclear titration of muscleblind 1 protein by exogenous CAG repeat expansions expressed in COSM6 cells, 22 by SCA3 RNA mutation in Drosophila 23 and by untranslated CAG expansion in transgenic Caenorhabditis elegans 24 and transgenic mice. bidirectional transcription through the repeat region results in an overlap of at least two mechanisms: toxic protein gain-of-function and toxic RNA gain-of-function.…”

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

“…repeat RNAs bind to and co-localize with the muscleblind (MBNL1) protein 12 . Originally MBNL1 had been found to bind to CUG or CCUG repeat mRNA-hairpin structures in patients with myotonic dystrophy (MD).…”

“…CAG repeat RNAs fold into secondary hairpin structures [9][10][11][12] . To give an idea of how these hairpins could look like, we performed in silico RNA-structure predictions of CAG repeat stretches with different repeat sizes.…”

“…CAG sequences form double-stranded RNA structures, the stability of which increases with repeat-numbers [9][10][11] . For example the CAG repeat region of the HTT messenger RNA (mRNA) forms double-stranded hairpin structures 12 . Some proteins bind to such CAG structures in a repeat size-dependent manner 13 .…”

“…Some proteins bind to such CAG structures in a repeat size-dependent manner 13 . Thus, it is thought that expansion of CAG repeat structures leads to the formation of pathological RNA-protein complexes, which cause damage in both-the nucleus where they influence the splicing machinery-and in the cytosol where they interfere with mRNA processing and translation 12,14 .…”