Connecting sulfur transfer pathways for molybdenum cofactor biosynthesis and tRNA thiolation in humans

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Sulfur is an essential element for all living organisms. The trafficking and delivery of sulfur to the molybdenum cofactor and nucleosides in tRNA is a highly regulated process which proceeds in a complex pathway involving several sulfur transferring proteins. Still numerous open questions remain in connecting both pathways in humans, which we want to study in this project in more detail. In humans, the cytosolic sulfur modification of mcm5s2U34 in tRNAsLys, Gln, Glu requires the proteins NFS1, TUM1, MOCS3, URM1, CTU1 and CTU2. In contrast, the sulfur insertion for tm5s2U34 modifications in mitochondrial tRNALys, Gln, Glu involves the proteins NFS1, TUM1 and MTU1. Further, for the synthesis of the dithiolene group of Moco, the proteins MOCS2A, MOCS3, TUM1 and NFS1 are involved, a reaction which occurs in the cytosol of humans. Conclusively, Moco biosynthesis and tRNA thiolation are connected in the cytosol and share the same sulfur delivery pathway proteins composed of NFS1, MOCS3 and possibly TUM1. To dissect the complex sulfur transfer pathways in the systems of mitochondrial and cytosolic tRNA thiolation and Moco biosynthesis, one goal is to analyze the role of two newly identified TUM1-isoforms for tm5s2U formation in mitochondria and the formation of mcm5s2U34 and Moco in the cytosol. The goal is to dissect the specificity of the sulfur transfer pathways in detail and to analyze whether different routes of sulfur relay systems exist for cytosolic tRNA thiolation and Moco biosynthesis. Further it is of interest to analyze whether differences between yeast and human MOCS3 exist. The usage of constructed CRISPR/Cas9 knock out cell lines (MOCS3, TUM1) and the functional complementation with the yeast Uba4 protein, the different TUM1 isoforms or MOCS3 fragments and variants will reveal the cellular role of each protein in detail. In total, our studies will dissect the role of tRNA thiolation and its connection to Moco biosynthesis in human cells in each compartment in detail.