Repeat-associated non-ATG (RAN) translation

Abstract

Microsatellite expansions cause more than 40 neurological disorders, including Huntington's disease, myotonic dystrophy, and C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). These repeat expansion mutations can produce repeat-associated non-ATG (RAN) proteins in all three reading frames, which accumulate in disease-relevant tissues. There has been considerable interest in RAN protein products and their downstream consequences, particularly for the dipeptide proteins found in C9ORF72 ALS/FTD. Understanding how RAN translation occurs, what cellular factors contribute to RAN protein accumulation, and how these proteins contribute to disease should lead to a better understanding of the basic mechanisms of gene expression and human disease.

Keywords: C9ORF72 ALS/FTD; Huntington's disease; RAN translation; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); mouse models; myotonic dystrophy; nucleocytoplasmic transport; spinocerebellar ataxia; translation; translation initiation; trinucleotide repeat disease.

Figure 1.

Translation initiation mechanisms. A, canonical translation initiation involves the binding of the 5′ mRNA cap (eIF4F consisting of eIF4G, eIF4E, eIF4A, and eIF4B) and mRNA poly(A) tail (PABP), unwinding of the mRNA by the helicase activity of eIF4A, and recruitment of 43S complex (eIF5, eIF3, eIF2, and 40S ribosomal subunit) followed by scanning of the mRNA 5′ UTR in a 5′ to 3′ direction by the engaged 43S complex. Recognition of the initiation codon results in the 48S initiation complex formation and displacement of several initiation factors. B, internal ribosome entry site initiation occurs in a cap-independent manner from multiple viral and cellular RNA sequences that involve the recruitment of the cellular 43S ribosomal complex to internal sites with the RNA by specific initiation translation factors (ITAFs). Depending upon the viral IRES group (I–IV), all, some, or none of the typical canonical translation factors, including the initiation codon, may be required for translation initiation. C, repeat-associated non-ATG translation initiation is a repeat-length–dependent process that allows for initiation at noncanonical codons either within or adjacent to the expanded repeat tract. Evidence from the FXTAS CGG repeats and some reports for G₄C₂ repeats support a requirement for 5′ mRNA cap, eIF4E, and eIF4A suggesting cap-dependent and scanning mechanisms. However, other reports support cap-independent translation initiation mechanisms more similar to IRES initiation. The identity and requirement of other cellular initiation factors involved in RAN translation have yet to be determined.

Figure 2.

Pathogenesis of a microsatellite repeat expansion disorder. An illustration of the three nonexclusive disease mechanisms proposed for most microsatellite expansion disorders, using C9ORF72 ALS/FTD as an example, is shown. A, microsatellite repeat expansion mutation (G₄C₂·G₂C₄ for C9-ALS/FTD) results in transcriptional inhibition and/or epigenetic silencing that reduces the levels of the resulting protein product (75, 82, 83). B, expansion mutations produce up to six toxic RAN proteins from both sense and antisense mutant transcripts. These proteins disrupt normal cellular functions (e.g. nucleocytoplasmic transport) and/or overwhelm cellular coping mechanisms (e.g. protein homeostasis). In C9-ALS/FTD protein GOF effects lead to nucleolar dysfunction, ER stress, altered autophagy, cell to cell transmission of RAN proteins, nucleocytoplasmic transport, and nuclear envelope deficits (33–36). C, expansion RNAs sequester RBPs into nuclear foci reducing RBPs' availability and decreasing its normal function. Expansion transcripts may also interact with and disrupt the function of other cellular components, such as proteins of nuclear pore complex (86). Although the identity and altered function of the RBP protein for C9ORF72 hexanucleotide repeats are the subject of much debate (83, 96, 108, 110, 113–115), RNA GOF effects are well established for DM1 CUG repeats that sequester MBNL proteins (79, 84–88). Different therapeutic approaches (purple boxes) target the various expansion RNA and protein products. ASOs and small molecules (SM) have been used to target either the sense or antisense expansion RNAs, although the effect on the opposite strand is unclear. Alternatively, ASOs can target transcription of the expanded repeat, e.g. SUPT5H (170). Additionally, therapeutic approaches, including small molecules, have been aimed at the downstream consequences of the expansion mutations, such as increasing or improving protein clearance mechanisms. Antibodies against RAN proteins (188) (Ab) or overexpressing proteins involved in autophagy (190) are also therapeutic approaches.