Post transcriptional modifications are an invariant feature of RNA molecules and the pseudouridine (Y) isomer of uracil is the most abundant modification found. Although structural studies suggest that Y contributes to the stability of RNAs, the importance of this and other potential roles in vivo remain largely unknown. In the ribosomal (r) RNAs, Y is found in the critical functional centers, suggesting an important role for Y in ribosome function. Consistent with this, rRNA pseudouridylation is essential in eukaryotes. In contrast, it is dispensable in a model archaeon while the PI?s laboratory has demonstrated that in E. coli, loss of all rRNA Y modifications has only modest effects on growth, ribosome function and all other tested phenotypes. The location and high conservation of Y modifications in rRNAs contrasts with their dispensability in model bacteria and archaea and provides the impetus for us to determine Y function. The long-range goal of the proposal is to determine the function of Y in ribosomes of diverse bacteria. This goal will be pursued in 3 complementary Specific Aims:
Specific Aim 1 will use gene interaction approaches to uncover the functions of Y in E. coli. Preliminary results link Y with ribosome biogenesis and turnover, and these findings will be pursued by examining rRNA metabolism, ribosome assembly and translation activity in Y-deficient cells.
Specific Aim 2 will examine two of the known effects of Y loss in bacteria, a decreased efficiency of translation termination and sensitivity to hyperosmotic stress. Genetic and physiological analyses will be used to examine these two consequences of Y loss in wild type and Y-deficient cells.
Specific Aim 3 will determine if the E. coli result can be extended to other bacteria by testing if Y is also dispensable in Thermus thermophilus, a thermophilic bacterium, by deleting the Y synthases and characterizing the resulting mutants. The health relevance of the proposal derives from the association of impaired pseudouridylation with dyskeratosis congenita, a complex bone marrow failure syndrome in humans, as well as the contribution of Y to the toxicity of 5-fluorouracil, an anti-cancer therapy and the involvement of Y in rRNA folding and ribosome assembly. Given the differing routes for Y incorporation into rRNA in bacteria and eukarya, elucidation of the role of Y in ribosome biogenesis may facilitate the development of antibacterials that inhibit this essential bacterial function.
Post-transcriptional modifications are found in all classes of RNA molecules, including those that make up the protein synthesis systems, yet their functions remain unclear. In this proposal, we investigate the function of the most abundant modification, pseudouridine (Y), by characterizing bacterial mutants lacking it. Uncovering Y functions can lead to a better understanding of their involvement in human disease and also may aid the exploitation of ribosome assembly as a target for antibacterial development.