Repeat-Associated Non-ATG (RAN) Translation in Fuchs' Endothelial Corneal Dystrophy

Abstract

PurposeThe strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD.MethodsAntibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF.ResultsCTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium.ConclusionsExpanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.

Towards Clinical Trials in Fuchs Endothelial Corneal Dystrophy: Classification and Outcome Measures—The Bowman Club Lecture 2019

“…This has led to the possibility of halting or slowing the progression of the disease by using targeted molecular therapies, such as to block RNA toxicity 27. The recognition of other downstream mechanisms in FECD, including repeat associated non-ATG translation,28 the unfolded protein response29 and oxidative stress,30 may similarly enable novel targeted therapies. The ability to culture human corneal endothelial cells ex vivo 31 has already led to promising clinical trials of cultured cell injection therapy, merging advances in basic science with advances in surgical approaches 31 32.…”

Repeat-associated non-ATG (RAN) translation

“…The CTG expansion mutation results in RNA foci and RNA splicing defects in endothelial cells from FECD patients, supporting an RNA GOF mechanism for this disease (72,73). Soragni et al (70) recently demonstrated that the intronic CTG⅐CAG expansion in TCF4 undergoes RAN translation in transfected cells and that the resulting antisense poly(Ser) and poly(Gln) RAN proteins are toxic to immortalized corneal endothelial cells. Additionally, the authors developed a C-terminal antibody against the sense poly(Cys) expansion protein expressed from the TCF4 CUG expansion RNAs and provided evidence for the accumulation of a poly(Cys) protein in patients' endothelial samples (70).…”

“…The most recent repeat expansion disorder shown to express RAN proteins is FECD (70). FECD involves the slowly-progressive degeneration of the corneal endothelium, which ultimately results in vision loss.…”

“…Soragni et al (70) recently demonstrated that the intronic CTG⅐CAG expansion in TCF4 undergoes RAN translation in transfected cells and that the resulting antisense poly(Ser) and poly(Gln) RAN proteins are toxic to immortalized corneal endothelial cells. Additionally, the authors developed a C-terminal antibody against the sense poly(Cys) expansion protein expressed from the TCF4 CUG expansion RNAs and provided evidence for the accumulation of a poly(Cys) protein in patients' endothelial samples (70). These data provide another opportunity to understand the mechanisms of RAN translation and the tissuespecific pathogenic consequences of the protein products of a repeat expansion.…”

RAN translation of the expanded CAG repeats in the SCA3 disease context

“…These expanded repeat tracts, located mainly in the non-coding regions of specific genes, can trigger noncanonical translation, i.e., in the absence of an AUG initiation codon induce the synthesis of various sets of potentially toxic proteins composed of homopolymeric, dipeptide, tetrapeptide or even pentapeptide repeated tracts of amino acids. RAN translation can be initiated in multiple reading frames in both sense and antisense directions, leading to the expression of distinct proteins [10][11][12][13][14][15][16][17][18][19]. The precise mechanism of RAN translation initiation is still poorly understood; however, it has been shown that the repeat tract length and RNA structure formed by repeats play crucial roles [20][21][22][23].…”