Cancer cells typically show an increase in protein synthesis. tRNAs are RNA molecules that play a central role in protein synthesis, directly base-pairing with the mRNA codons. A subset of tRNAs with uridine in position 34 (U34) must be modified to mcm5s2U34 to base pair efficiently with the corresponding A-ending codons. Synthesis of mcm5s2U34 is a multistep process, in which the first to act is the Elongator complex (ELP1-6), with ELP3 as catalytic subunits. We have previously shown that ELP3 is over-expressed in prostate cancer (PCa) [1] and here we present evidence of the relevance of mcm5s2U34 tRNA modification pathway, highlighting the effect on cellular metabolism and protein synthesis.
Using CRISPR/Cas9 technology, we depleted ELP3 in PCa cell lines DU 145, LNCaP, BM67 and untransformed prostate epithelial cell line PNT1A. Depletion of ELP3 strongly reduces the viability of PCa cell lines, but has no effect on PNT1A. Polysome profiling of ELP3 KO DU145 revealed a prominent downregulation of the global level of protein synthesis, confirmed by the analysis of protein mass-spectrometry data. Protein mass spectrometry further highlighted the impairment of key signalling pathways. Western blot validation showed that, upon ELP3 depletion, phosphorylation of AKT is strongly reduced, while eIF2alpha is potently phosphorylated with a parallel induction of ATF4. Bioinformatic analysis of the codon usage in the proteomic datasets shows that A-ending codons are used far less in DU 145 ELP3 KO cell lines.
Our results suggest that lack of U34 modification greatly impacts mRNA translation in a codon-specific manner, reducing the synthesis of proteins enriched in A-ending codons. In turn, this leads to global downregulation of protein synthesis, mediated by the activation of key signalling pathways. Our data points out that lack of mcm5s2U34 modification affects only neoplastic cells, making it an interesting novel target.