Publication Date:
2021-04-15
Description:
Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but 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 LPAC and QAGR RAN protein levels are reduced in PKR−/− and 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 both non-AUG and close-cognate initiation occurs 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 is 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.
Print ISSN:
0964-6906
Electronic ISSN:
1460-2083
Topics:
Biology
,
Medicine
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