Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase

Nucleic Acids Res. 2018 Jan 9;46(1):e1. doi: 10.1093/nar/gkx867.


Cellular accumulation of repetitive RNA occurs in several dominantly-inherited genetic disorders. Expanded CUG, CCUG or GGGGCC repeats are expressed in myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), or familial amyotrophic lateral sclerosis, respectively. Expanded repeat RNAs (ER-RNAs) exert a toxic gain-of-function and are prime therapeutic targets in these diseases. However, efforts to quantify ER-RNA levels or monitor knockdown are confounded by stable structure and heterogeneity of the ER-RNA tract and background signal from non-expanded repeats. Here, we used a thermostable group II intron reverse transcriptase (TGIRT-III) to convert ER-RNA to cDNA, followed by quantification on slot blots. We found that TGIRT-III was capable of reverse transcription (RTn) on enzymatically synthesized ER-RNAs. By using conditions that limit cDNA synthesis from off-target sequences, we observed hybridization signals on cDNA slot blots from DM1 and DM2 muscle samples but not from healthy controls. In transgenic mouse models of DM1 the cDNA slot blots accurately reflected the differences of ER-RNA expression across different transgenic lines, and showed therapeutic reductions in skeletal and cardiac muscle, accompanied by improvements of the DM1-associated splicing defects. TGIRT-III was also active on CCCCGG- and GGGGCC-repeats, suggesting that ER-RNA analysis is feasible for several repeat expansion disorders.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyotrophic Lateral Sclerosis / genetics*
  • Amyotrophic Lateral Sclerosis / metabolism
  • Animals
  • Base Sequence
  • Electrophoresis, Capillary
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Humans
  • Introns / genetics*
  • Mice, Transgenic
  • Muscle, Skeletal / metabolism
  • Muscle, Skeletal / pathology
  • Myotonic Dystrophy / genetics*
  • Myotonic Dystrophy / metabolism
  • RNA / genetics*
  • RNA Splicing
  • RNA-Directed DNA Polymerase / genetics*
  • RNA-Directed DNA Polymerase / metabolism
  • Repetitive Sequences, Nucleic Acid / genetics*
  • Temperature


  • RNA
  • RNA-Directed DNA Polymerase

Supplementary concepts

  • Amyotrophic lateral sclerosis 1