Protein Drg1 in baker´s yeast

The yeast Saccaromyces cerevisiae is an excellent model for the study of the life cycle of mRNA molecules. After mRNAs are formed in the nucleus by transcription they are processed in the same cellular compartment by cleavage at the 3`-end, the addition of a poly (A)- tail to the newly formed ends and the attachment of the cap structure at the 5`-end. The mature mRNA molecules are transported as RNP-particles through the nuclear pores into the cytoplasm where they are attached to the components of the translation machinery. In the cytoplasm the mRNA molecules are monitored for their correct composition by a quality control system, the NMD pathway. This system eliminates aberrant mRNA molecules by exposing them to an endonuclease. We have found that treatment of yeast cells with the drug diazaborine results in the occurrence of aberrant mRNA molecules. These aberrant mRNAs were absent when a yeast mutant which carries the DRG1-1 allele was treated with diazaborine. Gene DRG1 encodes a type II AAA-protein with an as yet unknown function. The allele DRG1-1 mediates diazaborine resistant growth characteristics. When we tested for other proteins which interact with protein Drg1 using the "Yeast Two Hybrid System" we identified four different proteins of the nuclear pore complex. In the present project application we propose to investigate the effect of the protein Drg1 on the maturation of mRNAs and their nucleo-cytoplasmic transport. Our working hypothesis specifies that diazaborine blocks the dissociation of the RNP-particle into its components and thus prevents the action of the quality control system. We also hypothesize that protein Drg1 in conjunction with the nuclear pore proteins helps to unfold the RNP-particles. We wish to test these hypotheses by investigating the possible interaction of yeast proteins that participate in mRNA metabolism and transport with protein Drg1. Particular attention will be paid on proteins of the RNP-particle and the nuclear pore. Furthermore, we will test the effects of mutants in genes of the NMD quality control system. We will investigate these effects by synthetic lethality testing and by using the yeast two hybrid system. GFP fusion constructs will also be included in this studies. Besides protein interaction studies we will also investigate the effect of various mutants on mRNA metabolism by northern blotting and polysome analyses. No aberrant molecules were observed in YAP1-specific mRNA when it was isolated from diazaborine treated yeast. We will explore the possibility that this is due to the presence of an uORF preceding the structural gene. Another part of the project will focus on the mechanism of diazaborine resistance mediated by alleles of gene DRG1. Preliminary experiments have shown that the mRNA of two resistant alleles is very stable. The mechanism of this stability will be explored.

The antibacterially active drug diazaborine was found to inhibit eukaryotic organisms as well, as exemplified with the single celled fungus Saccharomyces cerevisiae, the baker`s yeast. The work in the project centred at the question concerning the mechanism of action of the drug. Diazaborine resistant strains showed that mutations in a gene called DRG1, alias AFG2, mediate high level drug resistance. Hence, the work was focused next to the activity of diazaborine also on the function of this gene. The direct effect of diazaborine on wild type yeast cells showed a sharp drop in the number of large ribosomal subunits. Follow up studies showed that the complex sequence of the nucleolytic processing cascade from the primary transcript to the mature 25S ribosomal RNA was inhibited at a specific step that could be identified. The function of the protein Drg1, which is coded by gene DRG1, was investigated using thermosensitive drg1 mutants. When these mutants were grown at the nonpermissive temperature again a sharp drop in the number of large ribosomal particles was observed. Closer investigation into the formation of the ribosomal RNA showed again a block in the processing cascade, albeit at a different step as that observed in diazaborine treated cells. However, since protein Drg1 occurs exclusively in the cytoplasm and the rRNA processing is a nuclear event it is necessary to explain how components of these two cellular compartments interact to bring about the effects which we observed. We believe that a nuclear factor accompanies the preribosomal particle en route from the nucleus to the cytoplasm and, after being liberated by Drg1, recycles back into the nucleus. This step may be inhibited in the thermosensitive mutant. We believe to have found a candidate of such a shuttling protein recently which is presently being tested in support of our theory. Yeast strains carrying resistant alleles of DRG1 may make ribosomes with slightly altered functions which may be observed in particular situations. The literature describes the high level appearance of an endogenous double stranded RNA virus, called L-A, which is present in most laboratory strains at low level, when the cell produces altered ribosomes. We have observed the appearance of this virus in certain DRG1- mutants. This may be an indication of the altered nature of such ribosomes. The work of this project is important for the understanding of the novel gene DRG1 and may be relevant for the development of new antifungals or inhibitors of other eukaryotic cells as it identified a novel drug target.