Repeat associated non-ATG (RAN) translation: new starts in microsatellite expansion disorders

Abstract

Microsatellite-expansion diseases are a class of neurological and neuromuscular disorders caused by the expansion of short stretches of repetitive DNA (e.g. GGGGCC, CAG, CTG …) within the human genome. Since their discovery 20 years ago, research into how microsatellites expansions cause disease has been examined using the model that these genes are expressed in one direction and that expansion mutations only encode proteins when located in an ATG-initiated open reading frame. The fact that these mutations are often bidirectionally transcribed combined with the recent discovery of repeat associated non-ATG (RAN) translation provides new perspectives on how these expansion mutations are expressed and impact disease. Two expansion transcripts and a set of unexpected RAN proteins must now be considered for both coding and 'non-coding' expansion disorders. RAN proteins have been reported in a growing number of diseases, including spinocerebellar ataxia type 8 (SCA8), myotonic dystrophy type 1 (DM1), Fragile-X tremor ataxia syndrome (FXTAS), and C9ORF72 amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD).

Figure 1. The discovery of RAN translation in SCA8

(A) Bidirectional transcription at the SCA8 locus produces CUG expansion transcripts that form RNA foci and CAG expansion transcripts that produce a short ATG-initiated poly-glutamine expansion protein [10]. (B) Surprisingly, mutating the only ATG initiation-codon upstream of the CAG repeat did not prevent the expression of the poly-glutamine protein [12]. (C) Protein blot showing repeat expansion proteins detected by epitope tags are expressed from all three reading frames (poly-glutamine, poly-alanine and poly-serine) without an ATG-initiation codon. Expression of these repeat-associated non-ATG (RAN) proteins is repeat-length dependent, with simultaneous expression from multiple reading frames observed from longer repeat tracts [12]. (D) Mass-spectrometry of the poly-Alanine protein was performed on cell lysates transfected with a modified epitope-tagged CAG_EXP construct that encoded an arginine interruption within the polyAla protein to allow trypsin digestion. MS and RNA analysis confirmed that polyAla proteins are expressed without an AUG initiation codon and identified a series of peptides that suggest translation initiation may occur in the polyAla frame at sites throughout the repeat tract [12].

Figure 2. One repeat - multiple RNA and protein products

Schematic diagram showing potentially toxic RNA and protein products expressed from a repeat expansion mutation through a combination of bidirectional transcription, ATG-initiated and repeat associated non-ATG (RAN) translation. In vitro studies predict ATG-initiated and RAN translation can both occur when the repeat is located in an open reading frame (ORF) [12]. While a single ATG-initiated protein is illustrated, multiple ATG-initiated proteins may be produced if there are multiple ORFs. Additionally, RAN translation of the expanded repeat results in the expression of up to six distinct RAN proteins. For example, a CTG•CAG expansion can produce poly-Gln, poly-Ala and poly-Ser RAN proteins from the CAG transcript and poly-Leu, poly-Ala and poly-Cys RAN proteins from the CUG transcript. Each RAN protein, depending upon flanking sequences, may contain distinct C-terminal regions and an ATG-initiated protein in the same reading frame may also have a distinct N-terminal region.

Figure 3. Triple threat - three disease mechanisms for microsatellite expansion disorders

Expanded microsatellite repeats have been traditionally classified as either coding disorders or non-coding disorders that give rise to protein gain- or loss-of-function or RNA toxicity mechanisms. For traditional “coding” disorders, the repeat expansion is translated as part of a larger open-reading frame (ORF) and results in the expression of a mutant protein that disrupts normal cellular function and induces toxicity. For example Huntington’s disease (HD), a late-onset neurodegenerative disorder, is caused by a CAG expansion within the first exon of huntingtin gene that is translated as a polyglutamine tract in the huntingtin protein, HTT [78]. For traditional “non-coding” disorders (blue), the repeat expansion remains in the RNA transcript, accumulates as RNA foci that sequester RNA-binding proteins and lead to a loss of their normal function. For example, in myotonic dystrophy, CUG(G) expanded RNA transcripts sequester MBNL proteins from their normal splicing targets leading to a MBNL loss-of-function and alternative splicing dysregulation [, –81]. The recent discovery of repeat associated non-ATG (RAN) translation [12] adds a third pathway for disease. RNA transcripts from both “non-coding” and “coding” disorders may undergo RAN translation. Once in the cytoplasm, these transcripts are capable of producing proteins in all three reading frames, which may contribute to cellular toxicity/stress. Depending upon the flanking sequences, each of these RAN proteins will have a distinct expanded peptide repeats (colored boxes) and unique different C-terminal regions (f1, f2 and f3). If the repeat is also within an ATG-initiated open-reading frame, this ATG-initiated protein will share the expanded peptide repeat and C-terminal region with one of the RAN proteins but will have an additional N-terminal region. Further complexity is added by fact that many expansion mutations are bidirectionally transcribed [2], which doubles the number of distinct RAN proteins that may be produced. While individual RAN proteins have been observed in SCA8[12],DM1[12] and FXTAS[24] patients, sense and antisense RNA foci and RAN proteins in all six reading frames been shown to accumulate in C9ORF72 ALS/FTD patient cells [–, –48].