Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA
- 1University of Michigan
Abstract
A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is the most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). This element serves as a template for repeat-associated non-AUG (RAN) translation, which generates neurotoxic dipeptide repeat (DPR) proteins that aggregate in patient brains and contribute to neurodegeneration in model systems. To understand the mechanisms underlying RAN translation initiation, we used cryo-electron microscopy to determine the structure of an in vitro assembled human late-stage C9orf72 translation initiation complex at 3.2 A resolution and a near-native complex purified from human cell lysate at 3.5 A resolution. RAN translation on C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located 24 nucleotides upstream of the G4C2 repeat and is flanked by a strong endogenous Kozak sequence. The structures further reveal a direct interaction between the G4C2 repeat and 18S ribosomal RNA expansion segment 9 (ES9S) on the 40S small ribosomal subunit through a kissing-loop interaction. Preventing this RNA-RNA interaction using complementary antisense oligonucleotides significantly reduces RAN translation in vitro and in patient-derived iNeurons, indicating that this structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation and highlight a novel potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative disease.
Related
- Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein.
- Multi-level repression ensures tight translation control of C9ORF72
- Evolutionary extensions of IF2 couple translation initiation to bacterial stress adaptation
- Translation initiation by the Kozak mRNA sequence is based on a conformational readout on the ribosome
- m1A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation
BibTeX
@misc{kaufhold2026structural,
title = {{Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA}},
author = {R. Kaufhold and W. Dong and I. Purwar and B. Saladin and C. M. Wieland and A. Krans and E. Laimon and I. Malik and S. Leskaj and T. Havilio and M. Sokabe and C. S. Fraser and P. K. Todd and J. Brito Querido},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.25.761709},
url = {https://www.biorxiv.org/content/10.64898/2026.10.25.761709v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA
AU - R. Kaufhold
AU - W. Dong
AU - I. Purwar
AU - B. Saladin
AU - C. M. Wieland
AU - A. Krans
AU - E. Laimon
AU - I. Malik
AU - S. Leskaj
AU - T. Havilio
AU - M. Sokabe
AU - C. S. Fraser
AU - P. K. Todd
AU - J. Brito Querido
PY - 2026
DA - 2026/10/25
PB - bioRxiv
DO - 10.64898/2026.10.25.761709
UR - https://www.biorxiv.org/content/10.64898/2026.10.25.761709v1
AB - A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is the most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). This element serves as a template for repeat-associated non-AUG (RAN) translation, which generates neurotoxic dipeptide repeat (DPR) proteins that aggregate in patient brains and contribute to neurodegeneration in model systems. To understand the mechanisms underlying RAN translation initiation, we used cryo-electron microscopy to determine the structure of an in vitro assembled human late-stage C9orf72 translation initiation complex at 3.2 A resolution and a near-native complex purified from human cell lysate at 3.5 A resolution. RAN translation on C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located 24 nucleotides upstream of the G4C2 repeat and is flanked by a strong endogenous Kozak sequence. The structures further reveal a direct interaction between the G4C2 repeat and 18S ribosomal RNA expansion segment 9 (ES9S) on the 40S small ribosomal subunit through a kissing-loop interaction. Preventing this RNA-RNA interaction using complementary antisense oligonucleotides significantly reduces RAN translation in vitro and in patient-derived iNeurons, indicating that this structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation and highlight a novel potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative disease.
SN - 2692-8205
ER -