Periodic Reporting for period 4 - CODEKILLER (Killer plasmids as drivers of genetic code changes during yeast evolution)
Okres sprawozdawczy: 2023-04-01 do 2025-03-31
The aim of the CODEKILLER project was to investigate a possible mechanism by which the genetic code can change during evolution. The genetic code is the set of rules that determine how the 64 different codons in mRNA are translated into the 20 different amino acids in protein molecules, for example the codon AUG is translated into methionine. In the cell, the genetic code is physically implemented by tRNA molecules, which are covalently attached to an amino acid at one end and make basepairs with codons in mRNA at the other end. The genetic code is often considered to be universal because it is the same in the nuclear genomes of most species, but in 2018 we discovered that the genetic code has changed several times during the evolution of budding yeast species. Specifically, the codon CUG, which is translated as leucine in most species, is translated as serine or alanine in some groups of yeasts. Because this change is almost the only evolutionary change in the genetic code that has ever happened during the evolution of nuclear genomes, our goal was to understand how it happened. Our hypothesis was that the evolutionary reassignment of the codon CUG was caused by natural selection imposed by a killer toxin, specifically a ribonuclease toxin that cleaves and destroys tRNA-Leu molecules with the anticodon CAG, which translate CUG codons as leucine. We postulated that many different killer toxins that cleave specific tRNAs may exist in yeasts, even though such toxins are rare and only 4 of them were known at the outset of the CODEKILLER project.
By a combination of data-mining, targeted genome sequencing, and advanced bioinformatics methods, we increased the number of known toxins from 4 to more than 100. We found that killer toxins of this type are much older and present in a much broader range of fungal species than previously known. We found them in filamentous fungi and “early diverging” (basal) fungi as well as budding yeasts. We found that toxin genes can be located either in the nuclear genome, or on small virus-like elements in the cell’s cytoplasm.
We showed that many of the new toxins we discovered are functional, and we identified the tRNA targets of new toxins by using ribosome profiling, a new application of this technology. We found that toxin proteins evolve very quickly, and that they can change their tRNA targets during evolution. We also found that the corresponding immunity proteins evolve quickly.
Our results show that this class of tRNA-cleaving killer toxin pre-dates the genetic code changes in budding yeasts, so they are consistent with the hypothesis that the reassignment of the CUG codon was driven by a tRNA-cleaving killer toxin, although we did not find a toxin that specifically cleaves tRNA-Leu(CAG).
Results from the project were published as open-access papers in international peer-reviewed papers during 2022-2025.
We showed that many of the new toxins we discovered are functional, and we identified the tRNA targets of new toxins by using ribosome profiling, a new application of this technology. We found that toxin proteins evolve very quickly, and that they can change their tRNA targets during evolution. We also found that the corresponding immunity proteins evolve quickly.
Our results show that this class of tRNA-cleaving killer toxin pre-dates the genetic code changes in budding yeasts, so they are consistent with the hypothesis that the reassignment of the CUG codon was driven by a tRNA-cleaving killer toxin, although we did not find a toxin that specifically cleaves tRNA-Leu(CAG).
Results from the project were published as open-access papers in international peer-reviewed papers during 2022-2025.
The results obtained have greatly expanded our knowledge of the tRNA-cleaving (anticodon nuclease) family of killer toxins, and showed that this family is highly heterogeneous and evolving very rapidly. Quite unexpectedly, we found that similar killer toxins are also made by filamentous fungi such as Fusarium, where the toxins and their immunity factors are encoded by small clusters of genes in the nuclear genome. We also found that cytosolic plasmids, similar to the cytosolic plasmids that encode killer toxins in budding yeasts, are present in basal fungi such as Coemansia species. Our results have given the field a much clearer understanding of the ubiquity of this class of killer toxin, and its role in the evolution of the genetic code.