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

Solaleh Khoramian Tusi, Lien Nguyen, Kiruphagaran Thangaraju, Jian Li, John D Cleary, Tao Zu, Laura P W Ranum

Human molecular genetics 2021 May 31

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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. © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Citation

Solaleh Khoramian Tusi, Lien Nguyen, Kiruphagaran Thangaraju, Jian Li, John D Cleary, Tao Zu, Laura P W Ranum. The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats. Human molecular genetics. 2021 May 31;30(11):1020-1029