Externally funded project

Functional cross-talk among modifications in the tRNA anticodon stem-loop (tRNA Mods II)


Project Details
Project duration: 07/201712/2018


Abstract
Although tRNAs undergo many chemically different modifications, their contributions to tRNA functioning during translation and protein synthesis are ill-defined and only beginning to emerge. To this end, we have identified tRNA anticodon stem loop modifications (mcm5s2U [5-methoxy-carbonyl-methyl-2-thiouridine], Psi [pseudo-uridine] and ct6A [cyclic N(6)-threonyl-carbamoyl-adenosine]) which are crucial for proper function of two specific tRNAs (tRNAGlnUUG or tRNALysUUU). In order to elucidate how these modifications cross-talk and impact on individual tRNA properties and decoding functions on a proteomic scale, we will achieve the following interlinked aims: - study the impact of combined anticodon modification defects on tRNA charging, abundance, integrity and/or decoding fidelity - compare on a proteome-wide scale via SILAC-MS/MS translational consequences between improper tRNA modification and tRNA cleavage by anticodon targeting tRNase toxins - validate targets and translation of modification tunable transcripts (MoTTs) in Western blots and by tRNA overexpression suppression assays - compare codon composition with MoTT concept and examine whether gene synthesis and codon re-engineering can uncouple MoTT translation from tRNA modification control - correlate overlapping translationally deregulated targets with observed phenotypes in tRNA modification mutants by gene ontology analysis Collectively, this complementary work programme will provide a concerted basis to examine modification effects on tRNA function and test the MoTT concept (see above), which postulates that tRNA modifications might fine-tune the proteome by affecting mRNA translation in a codon usage dependent manner. We will seek for correlations between deregulated proteins under conditions of inactivating specific tRNAs (tRNAGlnUUG or tRNALysUUU) which lead to similar pleiotropic cellular defects. Eventually, sorting deregulated proteins into functional categories may uncover the mechanistic principle of how simultaneous loss of the above tRNA modifications induces such phenotypes. Together, these efforts may address how functional cross-talk among anticodon stem-loop modifications is able to protect against cellular dysfunctions, which in higher eukaryotes - including our own cells - can translate into formation of cancer or neuropathies as severe as ALS. Therefore, using budding yeast as a model system, we consider the project and its specific tRNA modification focus is logically linked to the topic of DFG SPP 1784 Chemical Biology of Native Nucleic Acid Modifications and has the potential to provide innovative mechanistic insights into the biological significance of tRNA anticodon modification pathways and their functional cross-talk.

Last updated on 2018-26-04 at 13:40