Abstract
Ribosome-associated quality control (RQC) relieves stalled ribosomes and eliminates potentially toxic nascent polypeptide chains (NCs) that can cause neurodegeneration. During RQC, RQC2 modifies NCs with a C-terminal alanine and threonine (CAT) tail. CAT tailing promotes ubiquitination of NCs for proteasomal degradation, while RQC failure in budding yeast disrupts proteostasis via CAT-tailed NC aggregation. However, the CAT tail and its cytotoxicity in mammals have remained largely uncharacterized. We demonstrate that NEMF, a mammalian RQC2 homolog, modifies translation products of nonstop mRNAs, major erroneous mRNAs in mammals, with a C-terminal tail mainly composed of alanine with several other amino acids. Overproduction of nonstop mRNAs induces NC aggregation and caspase-3-dependent apoptosis and impairs neuronal morphogenesis, which are ameliorated by NEMF depletion. Moreover, we found that homopolymeric alanine tailing at least partially accounts for CAT-tail cytotoxicity. These findings explain the cytotoxicity of CAT-tailed NCs and demonstrate physiological significance of RQC on proper neuronal morphogenesis and cell survival.
Highlights
Disturbance of protein synthesis affects a wide range of biological processes and could be the cause of various human diseases, including cancers and neurologic disorders
Nonstop Proteins Are C-terminal alanine and threonine (CAT) Tailed by NEMF Since the ribosome engaging a poly(A) tail of the nonstop mRNA produced by erroneous polyadenylation represents the primary Ribosome-associated quality control (RQC) targets in mammals (Akimitsu et al, 2007; Garzia et al, 2017), we examined whether nonstop mRNAs are targeted by the RQC pathway and the protein products are CAT tailed using b-globin and EGFP nonstop mRNAs (Figures 1A)
The level of nonstop products was increased upon LTN1 knockdown and reduced by wildtype (WT) LTN1 overexpression, but not by the mutant lacking the RING domain, a domain essential for E3 ubiquitin ligase activity (Figures 1B, 1C, S1A, and S1B), which confirms that the RQC pathway targets nonstop mRNAs in mammalian cells
Summary
Disturbance of protein synthesis affects a wide range of biological processes and could be the cause of various human diseases, including cancers and neurologic disorders. Mutations in certain RNA-binding proteins could affect mRNA synthesis, metabolism, and translational control of specific or bulk mRNAs, and these could be associated with neurodegeneration and mental retardation. The aberrant mRNAs spontaneously produced in cells by erroneous gene expression could affect translation, and their incomplete protein products could be toxic to cells. To prevent such deleterious effects of aberrant gene expression, cells have evolved various quality control pathways. One such pathway is ribosome-associated quality control (RQC)
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