Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2ß, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.

SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity.

Transcribed CGG repeat expansions cause neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic peptides. To identify proteins involved in these processes, we employed a CGG repeat RNA-tagging system to capture repeat-associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine-rich) proteins that interact selectively with CGG repeats basally and under cellular stress. These proteins modify toxicity in a Drosophila model of FXTAS. Pharmacologic inhibition of serine/arginine protein kinases (SRPKs), which alter SRSF protein phosphorylation, localization, and activity, directly inhibits RAN translation of CGG and GGGGCC repeats (associated with C9orf72 ALS/FTD) and triggers repeat RNA retention in the nucleus. Lowering SRPK expression suppressed toxicity in both FXTAS and C9orf72 ALS/FTD model flies, and SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. Together, these findings demonstrate roles for CGG repeat RNA binding proteins in RAN translation and repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders.