Talk Abstracts

Talk on Friday 03:30-03:45pm submitted by Brenna Saladin

Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA

Brenna Saladin (Department of Biological Chemistry, University of Michigan), Ross Kaufhold, Wenzhao Dong, Ishita Purwar, Suada Leskaj (Department of Biological Chemistry, University of Michigan), Clare Wieland, Amy Krans, Erinn Laimon, Indranil Malik (Department of Neurology, University of Michigan), Masaaki Sokabe, Christopher Fraser (Department of Molecular and Cellular Biology, University of California), Peter Todd (Department of Neurology, University of Michigan), Jailson Brito Querido (Department of Biological Chemistry, University of Michigan)

Abstract:
The most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) is a GGGGCC (G4C2) hexanucleotide repeat expansion within the first intron of C9orf72 [1,2]. This site serves as a template for repeat-associated non-AUG (RAN) translation, wherein translation initiates at a non-canonical start site (non-AUG) codon upstream or within an expanded repeat sequence [1,3]. RAN translation of the expanded G4C2 repeat results in production of toxic dipeptide repeat (DPR) proteins that aggregate in patient brains and have been shown to contribute to neurodegeneration in model systems [4]. 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 G4C2 C9orf72 translation initiation complex at 3.2 resolution and a near-native complex purified from human cell lysate at 3.5 resolution. RAN translation of the G4C2 C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located upstream of the G4C2 repeat and flanked by a strong endogenous Kozak sequence. The structures further reveal an interaction between the G4C2 repeat mRNA and 18S ribosomal RNA expansion segment 9 (ES9^s) on the 40S small ribosomal subunit through a kissing-loop interaction. Complimentary antisense oligonucleotides that prevent this mRNA-rRNA interaction significantly reduced RAN translation in patient-derived iNeurons, indicating that his structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation at the expanded G4C2 repeat in the C9orf72 gene, and highlight a potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative diseases.

References:
1. Malik, I., Kelley, C. P., Wang, E. T. & Todd, P. K. Molecular mechanisms underlying nucleotide repeat expansion disorders. Nat. Rev. Mol. Cell Biol. 22, 589607 (2021).
2. Balendra, R. & Isaacs, A. M. C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat. Rev. Neurol. 14, 544558 (2018).
3. Brito Querido, J., Daz-Lpez, I. & Ramakrishnan, V. The molecular basis of translation initiation and its regulation in eukaryotes. Nat. Rev. Mol. Cell Biol. 25, 168186 (2024).
4. Wen, X. et al. Antisense Proline-Arginine RAN Dipeptides Linked to C9ORF72-ALS/FTD Form Toxic Nuclear Aggregates that Initiate In Vitro and In Vivo Neuronal Death. Neuron 84, 12131225 (2014).

Keywords: RAN translation, translation initiation, neurodegeneration