The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats

Hum Mol Genet. 2021 May 31;30(11):1020-1029. doi: 10.1093/hmg/ddab098.


Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics
  • DNA Repeat Expansion / genetics
  • Eukaryotic Initiation Factor-2 / genetics*
  • HEK293 Cells
  • Humans
  • Microsatellite Repeats / genetics
  • Myotonic Dystrophy / genetics*
  • Myotonic Dystrophy / physiopathology
  • Protein Biosynthesis / genetics
  • eIF-2 Kinase / genetics*


  • Eukaryotic Initiation Factor-2
  • EIF2AK2 protein, human
  • EIF2AK3 protein, human
  • eIF-2 Kinase