Optimized protocol for tRNA identification in the ribosomal complexes from human cell lines.

Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers. For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021).

Failure to Degrade CAT-Tailed Proteins Disrupts Neuronal Morphogenesis and Cell Survival.

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.