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Abstract

All organisms, from bacteria to humans, possess dedicated molecular mechanisms to ensure accurate translation surveillance. Ribosome-associated quality control (RQC) is one such mechanism that eliminates truncated polypeptides arising from ribosomal stalling during translation. In canonical mammalian RQC, NEMF binds to large ribosomal subunits that are obstructed by nascent polypeptides, and recruits the E3 ubiquitin ligase Listerin to target these aberrant nascent chains for proteasomal degradation. Additionally, NEMF can add poly-alanine tails (Ala-tails) to the nascent polypeptide, which help expose buried lysine residues for Listerin-mediated ubiquitination but also act as degrons for E3 ligases KHLDC10 and Pirh2, providing a fail-safe mechanism. While mutations in Listerin or in NEMF are associated with neurodegeneration in mice and humans, the precise role of Ala-tailing in translational surveillance and its connection to neurodegeneration remain unclear.

Here, we analyzed the D96A mutation in NEMF, which selectively impairs Ala-tailing, in different model systems to decipher the physiological roles of this modification. In mice, homozygous D96A mutations caused only mild motor defects without affecting lifespan. However, in combination with the Listerin mutation, homozygous D96A exacerbated neurodegeneration, revealing a synthetic lethal phenotype. Interestingly, partial reduction of Ala-tailing alleviated Listerin mutation-induced neurodegeneration, suggesting that Ala-tailing can act as a double-edged sword, transitioning from neuroprotective to neurotoxic under specific conditions. Biochemical studies in HeLa cells indicate that Ala-tails promote amyloid-like aggregation of RQC substrates, thus providing a potential pathomechanism. Additional support comes from two other N-ethyl-N-nitrosourea (ENU)-induced mutations in mice: K489G, which disrupts Ala-tailing, has a similar phenotype to D96A homozygous mice; and R86S, which spares Ala-tailing but leads to severe neurodegenerative phenotypes, indicating that NEMF has broader functions. By developing a novel method to pull-down Ala-tailed proteins ("Pirhification"), I have also identified several RQC target proteins, thereby offering molecular insights into how defects in Ala-tailing may underlie neurodegenerative processes. Collectively, these findings highlight the importance of Ala-tailing in proteostasis and shed light on its molecular role in neurodegenerative pathophysiology.

To further investigate neuron-specific impacts of Ala-tailing and RQC, I employed an i-motor neuron (i-MN) model derived from induced pluripotent stem cells (iPSCs) and used CRISPR/Cas9 scarless gene editing to generate D96A-mutant iPSCs. Through a small-molecule differentiation protocol, I obtained high-purity i-MN cultures, and an RQC reporter system enabled molecular analyses. Here, I report that D96A i-MNs exhibited mild RQC defects alone but stabilized RQC substrates in a Listerin-mutant background, underscoring Ala-tailing’s essential role in neuronal function. Moreover, proteomic analysis revealed NEMF's involvement in gene networks linked to neuronal development and neurodegenerative diseases. Overall, this i-MN platform provides a powerful model to investigate RQC mechanisms and their implications in neurodegenerative disorders.