Computational investigation of RNA CUG repeats responsible for myotonic dystrophy 1.

Ilyas Yildirim,David J Wales,Debayan Chakraborty,Matthew D Disney,George C Schatz

doi:10.1021/acs.jctc.5b00728

Abstract

Myotonic Dystrophy 1 (DM1) is a genetic disease caused by expansion of CTG repeats in DNA. Once transcribed, these repeats form RNA hairpins with repeating 1×1 nucleotide UU internal loop motifs, r(CUG)n, which attract muscleblind-like 1 (MBNL1) protein leading to the disease. In DM1 CUG can be repeated thousands of times, so these structures are intractable to characterization using structural biology. However, inhibition of MBNL1-r(CUG)n binding requires a detailed analysis of the 1×1 UU internal loops. In this contribution we employ regular and umbrella sampling molecular dynamics (MD) simulations to describe the structural and thermodynamic properties of 1×1 UU internal loops. Calculations were run on a reported crystal structure and a designed system, which mimics an infinitely long RNA molecule with continuous CUG repeats. Two-dimensional (2D) potential of mean force (PMF) surfaces were created by umbrella sampling, and the discrete path sampling (DPS) method was utilized to investigate the energy landscape of 1×1 UU RNA internal loops, revealing that 1×1 UU base pairs are dynamic and strongly prefer the anti–anti conformation. Two 2D PMF surfaces were calculated for the 1×1 UU base pairs, revealing several local minima and three syn–anti ↔ anti–anti transformation pathways. Although at room temperature the syn–anti ↔ anti–anti transformation is not observed on the MD time scale, one of these pathways dominates the dynamics of the 1×1 UU base pairs in temperature jump MD simulations. This mechanism has now been treated successfully using the DPS approach. Our results suggest that local minima predicted by umbrella sampling calculations could be stabilized by small molecules, which is of great interest for future drug design. Furthermore, distorted GC/CG conformations may be important in understanding how MBNL1 binds to RNA CUG repeats. Hence we provide new insight into the dynamic roles of RNA loops and their contributions to presently incurable diseases.

Highlights

Repeat expansion disorders are caused by mutations in DNA where repeats in certain genes become expanded
One hypothesis is that formation of non-B-form conformations by the repetitive DNA sequences, such as hairpins, is the reason for the expansion during DNA replication and repair.[3−5] In the expansions of CAG repeats, which are typically found in the coding regions of mRNAs such as Huntingtin (HTT), androgen receptor (AR), spinocerebellar ataxia (SCA), and atrophin-1 (ATN1) genes, the transcripts are translated into toxic polyglutamine[6] proteins, resulting in Huntington’s disease (HD), Spinal and Bulbar Muscular Atrophy (SBMA), Spinocerebellar Ataxia Type 1 (SCA1), and Dentatorubral-pallidoluysian atrophy (DRPLA), respectively.[7−12] it has been shown recently that RNA CAG repeat expansions can sequester proteins and contribute to neurodegeneration.[13−15] Another genetic disorder, Friedreich’s ataxia (FRDA), is caused by expansion of GAA repeats.[9,16]
A common heritable form of mental retardation, Fragile X-associated tremor ataxia syndrome (FXTAS), is caused by expansion of the CGG segment from 50 to 200 repeats in the 5′-untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene.[7,16−23] Once the FMR1 gene is transcribed into mRNA, the expanded CGG repeats fold into a hairpin structure with repeating 1×1 GG internal loops that sequester and inactivate Sam[68] protein

Summary

Introduction

Repeat expansion disorders are caused by mutations in DNA where repeats in certain genes become expanded. One hypothesis is that formation of non-B-form conformations by the repetitive DNA sequences, such as hairpins, is the reason for the expansion during DNA replication and repair.[3−5] In the expansions of CAG repeats, which are typically found in the coding regions of mRNAs such as Huntingtin (HTT), androgen receptor (AR), spinocerebellar ataxia (SCA), and atrophin-1 (ATN1) genes, the transcripts are translated into toxic polyglutamine (polyQ)[6] proteins, resulting in Huntington’s disease (HD), Spinal and Bulbar Muscular Atrophy (SBMA), Spinocerebellar Ataxia Type 1 (SCA1), and Dentatorubral-pallidoluysian atrophy (DRPLA), respectively.[7−12] it has been shown recently that RNA CAG repeat expansions can sequester proteins and contribute to neurodegeneration.[13−15] Another genetic disorder, Friedreich’s ataxia (FRDA), is caused by expansion of GAA repeats.[9,16] A common heritable form of mental retardation, Fragile X-associated tremor ataxia syndrome (FXTAS), is caused by expansion of the CGG segment from 50 to 200 repeats in the 5′-untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene.[7,16−23] Once the FMR1 gene is transcribed into mRNA, the expanded CGG repeats fold into a hairpin structure with repeating 1×1 GG internal loops that sequester and inactivate Sam[68] protein.

Methods

Results

Conclusion