Mechanisms of repeat-associated non-AUG translation in neurological microsatellite expansion disorders

Abstract

Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases.

Keywords: RAN translation; microsatellite repeat expansion disorders; pathophysiology.

Figure 1.. Canonical and physiological translation initiation mechanisms.

(A) Canonical initiation involves the eIF4F complex and the poly(A) tail binding protein PABP binding to the mRNA and subsequently interacting with the 43S complex (eIF5, eIF3, eIF2 and the 40S ribosome) to form the 48S complex. eIF4E and PABP both interact with eIF4G to create a ‘closed loop complex'. eIF4A, with its cofactors eIF4B and eIF4H, interact with eIF4G and eIF4E to provide helicase activity to unwind secondary structures present in the 5′UTR. The 48S complex scans the mRNA for an AUG start codon, where the 60S ribosomal subunit is recruited through eIF5B and several of the initiator factors are displaced and recycled to initiate a new round of translation. (B) IRES mediated translation involves a strong secondary or tertiary structure within the 5′UTR. The precise mechanisms vary between viruses but the IRES element interacts with either the 40S subunit or eIF4G, which recruit any other required factors to initiate translation. (C) Canonical and alternative physiological initiator tRNA-binding eIF factors recognise different start codons. Canonical translation occurs through eIF2α, delivering Met-tRNA_i^Met to the P site of the 40S ribosomal subunit in a GTP-dependent manner, through interaction with both the canonical AUG start codon and near cognate start codons CUG and GCG. Both eIF2A and eIF2D are also able to initiate translation, however this can occur in a GTP-dependent or independent manner, binding either charged or uncharged tRNA_i^Met. eIF2A can additionally bind Leu-tRNA^CUG to initiate translation. eIF2A can initiate translation at AUG, CUG and UUG codons, while eIF2D can initiate at AUG, CUG, GCG and UUG codons.

Figure 2.. Known RNA structures and protein factors involved in RAN translation.

(A) RAN translation of FMR1 occurs in a Cap-, eIF4E- and eIF4A-dependent manner along with eIF4A cofactors eIF4H and eIF4B, recruiting the 40S ribosome and eIF2α-bound Met-tRNA_i^Met to the near-cognate ACG start codon upstream of the CGG repeat expansion. Regulation of start codon fidelity through eIF1 and eIF5 is important. Any potential role of the translation initiation factors PABP, eIF4G, eIF1A and eIF3 remain unknown. (B) RAN translation of the GGGGCC repeat expansion from C9ORF72 occurs in an eIF4A-dependent manner to recruit the 40S ribosome subunit and eIF2A-bound Met-tRNA_i^Met to the near cognate CUG start codon upstream of the hexanucleotide repeat expansion. The eIF4A cofactors eIF4B and eIF4H have been shown to be disease modifiers and are involved. Contradictory evidence for the role of the m⁷cap and eIF4E factor indicates that further elucidation of their roles is required. Any potential role of the translation initiation factors PABP, eIF4G, eIF1, eIF1A, eIF3 and eIF5 still remain unknown. The mechanisms involved in the RAN translation of C9ORF72 antisense CCCCGG-repeat transcripts have not yet been explored.

Figure 3.. The role of a uORF in the translation of RAN proteins from pathological sense GGGGCC C9ORF72 repeat expansions.

(A) In unstressed cellular conditions, a uORF of 55 nucleotides in length within intron 1 of C9ORF72 inhibits RAN translation of the downstream repeat expansion. The uORF is translated through the canonical translation machinery and ribosomes are unable to reassemble on the mRNA for initiation at the downstream CUG RAN initiation codon. (B) Following cellular stress, phosphorylation of eIF2α prevents its binding of Met-tRNA_i^Met results in an inhibition of eIF2-driven canonical translation. Consequently, the alternative tRNA recruiting factor eIF2A is able to initiate non-canonical translation. The scanning 40S ribosomal complex scans through the uORF and eIF2A initiates RAN translation at the downstream CUG codon.