Structural Diversity of Sense and Antisense RNA Hexanucleotide Repeats Associated with ALS and FTLD.

Tim Božič,Janez Plavec,Primož Šket,Boris Rogelj,Matja Zalar

doi:10.3390/molecules25030525

Abstract

The hexanucleotide expansion GGGGCC located in C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.

Highlights

The pathological hexanucleotide expansion GGGGCC located in the first intron or promoter region of the C9orf72 gene on chromosome 9p21 represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD)—two fatal neurodegenerative diseases with progressive loss of motor neurons in the brain and spinal cord [1,2,3,4,5,6,7]
By utilizing NMR spectroscopy and complementary methods we demonstrated that sense r(G4 C2 )4 and antisense r(G2 C4 )4 RNA oligonucleotides composed of four hexanucleotide repeats can form dimers and hairpins under conditions approaching physiological relevance
Dimeric nature of structure was identified based on translational diffusion coefficient (Dt ) value of 0.91 ± 0.05 × 10−10 m2 s−1 . 1D 1 H-NMR spectrum reveals six well resolved signals between δ 12.6 and 13.6 ppm corresponding to guanine imino protons involved in Watson-Crick base pairs (Figure 1b and Figure S1a)

Summary

Introduction

The pathological hexanucleotide expansion GGGGCC located in the first intron or promoter region of the C9orf gene on chromosome 9p21 represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD)—two fatal neurodegenerative diseases with progressive loss of motor neurons in the brain and spinal cord [1,2,3,4,5,6,7]. Healthy individuals possess up to 19 repeats, while patients with ALS or FTLD may carry up to 5000 repeats [3,4,5,6]. Three main non-exclusive mechanisms of expanded hexanucleotide G4 C2 repeats involved in ALS and FTLD have been proposed: protein C9orf haploinsufficiency, accumulation of dipeptide repeat proteins. Molecules 2020, 25, 525; doi:10.3390/molecules25030525 www.mdpi.com/journal/molecules (DPRs) and RNA toxicity [7]. While haploinsufficiency and DPRs are both protein driven mechanisms, RNA toxicity involves accumulating sense and antisense RNA transcripts. Accumulation leads to formation of RNA foci, which sequesters RNA-binding proteins [8]. Bound RNA-binding proteins are responsible for nuclear transport, splicing, and translation, all of which are affected by dysregulated

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Discussion

Conclusion