Near-cognate initiation generates FMRpolyG from CGG repeats in Fragile X associated Tremor Ataxia Syndrome

Abstract

Repeat associated non-AUG (RAN) translation of FMR1 5' UTR CGG repeats produces toxic homo-polymeric proteins that accumulate within ubiquitinated inclusions in Fragile X-associated tremor/ataxia syndrome (FXTAS) patient brains and model systems. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG codon located just 5' to the repeat. Methods to accurately measure FMRpolyG in FXTAS patients are lacking. Here we used data dependent acquisition (DDA) and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides (SIS) to identify potential signature FMRpolyG fragments in patient cells and tissues. Following immunoprecipitation (IP) enrichment, we detected FMRpolyG signature peptides by PRM in transfected cells, FXTAS human samples and patient derived stem cells, but not in controls. Surprisingly, we identified two amino-terminal peptides: one beginning with methionine (Ac-MEAPLPGGVR) initiating at an ACG, and a second beginning with threonine (Ac-TEAPLPGGVR), initiating at a GUG. Abundance of the threonine peptide was enhanced relative to the methionine peptide upon activation of the integrated stress response. In addition, loss of the eIF2 alternative factor, eIF2A, or enhanced expression of initiation factor eIF1, preferentially suppressed GUG initiated FMRpolyG synthesis. These data demonstrate that FMRpolyG is quantifiable in human samples and that RAN translation on FMR1 initiates at specific near cognate codons dependent on available initiation factors and cellular environment.

Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism

“…RAN translation of CGG repeats contributes to their toxicity in model systems (Green et al, 2017;Kearse et al, 2016;Sellier et al, 2017;Todd et al, 2013). The most-abundant CGG RAN product is a polyglycine protein, FMRpolyG, whose translation is initiated at three nearcognate codons-two GUGs and an ACG-upstream of the repeat (Kearse et al, 2016;Zhang et al, 2020). Similar to translation of other non-AUG-initiated genes, CGG RAN initiation is influenced by the availability of specific canonical initiation factors.…”

Partners in crime: Proteins implicated in RNA repeat expansion diseases

“…RNA with G4C2 repeats, but not antisense RNA with C4G2 repeats, formed nuclear foci in a length-dependent manner in vitro and in living cells (Jain & Vale, 2017). The observed difference may be associated with the secondary structures of those molecules, as the presence of G-quadruplexes promotes phase separation (Fay et al, 2017;Jain & Vale, 2017;Y. Zhang et al, 2020.…”

“…RNA with G4C2 repeats, but not antisense RNA with C4G2 repeats, formed nuclear foci in a length‐dependent manner in vitro and in living cells (Jain & Vale, 2017). The observed difference may be associated with the secondary structures of those molecules, as the presence of G‐quadruplexes promotes phase separation (Fay et al, 2017; Jain & Vale, 2017; Y. Zhang et al, 2020, Zhang et al, 2019). Moreover, foci driven by the expression of G4C2 exp RNA are less dynamic than CAG exp RNA foci but are similarly retained in nuclei and colocalize with nuclear speckles (Jain & Vale, 2017; Y.…”