Do chemical modifications of tRNAs contribute to the aggregation resilience of the highly Q-rich proteome in D. discoideum?

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The wobble uridines at position 34 (U34) in transfer RNAs (tRNAs) are chemically modified in eukaryotes by the Elongator complex. In the absence of these modifications, several severe phenotypes are observed. In yeast, these phenotypes can be attributed to distorted translation, which results in protein aggregation. Elevated levels of hypomodified tRNAs, however, can rescue these phenotypes.The amoeba Dictyostelium discoideum, the model organism of the evolutionary supergroup of Amoebozoa, features a proteome with exceptionally long stretches of the amino acids glutamine (Q) that in other organisms cause protein aggregation. Concurrent, it harbors a significantly increased copy number of tRNA genes that are modified by the elongator complex. We have generated strains in which the elongator-dependent modification pathway is disrupted at all major steps. Additionally, we have generated the first unconditional knockout of the tRNA gene of tQCUG in a eukaryotic species. Our analyses indicate that the chemical modifications introduced by the Elongator complex are conserved in the amoeba, but the structural composition deviates from that seen in yeast. Within this project, we will determine the structure of the Elongator complex of the amoeba at atomic resolution. D. dictyostelium can live in a unicellular or multicellular lifestyle. This offers the unique opportunity to compare the function of the Elongator complex in the unicellular and multicellular lifestyles. Using the generated mutant strains, we will quantitatively determine the exact chemical modifications that are present in either form of its life, and correlate this to phenotypic changes in the timing and progress of development.The unique tQCUG null strain, which we have generated, appears to depend on the presence of a functional Elongator complex, likely because the chemical modifications introduced in the isoacceptor tQUUG are important for decoding. We will generate a conditional knockout system, which will allow us to investigate whether the elongator-dependent modifications indeed are essential for the viability of the tQCUG null strain. Using reporter constructs, we will analyze, whether elongator mutants in the amoeba become susceptible to protein aggregation, in both the unicellular and the multicellular lifestyle. These investigations will be complemented by global studies on translation in these mutants, for which we will employ ribosome profiling. By these investigations, we want to unravel the contribution of chemical modifications on the resilience of the amoeba to protein aggregation. Understanding the mechanisms of how protein aggregates with their often devastating effects can be prevented should be important also for other species.