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

硫磺是所有生物的必需元素。硫向tRNA中的钼辅助因子和核苷的运输和运输是一个高度受调控的过程，涉及几个硫转移蛋白的复杂途径。在连接人类的两条通路方面，仍然有许多悬而未决的问题，我们希望在这个项目中更详细地研究这一点。在人类中，tRNAsLys、Gln、Glu中mcm5s2U34的胞浆硫修饰需要NFS1、TUM1、MOCS3、URM1、CTU1和CTU2蛋白。相反，线粒体tRNALys、Gln、Glu的tm5s2U34修饰的硫插入涉及蛋白质NFS1、TUM1和MTU1。此外，为了合成MOCO的二硫烯基，涉及到蛋白质MOCS2A、MOCS3、TUM1和NFS1，这一反应发生在人的细胞质中。综上所述，MOCO生物合成和tRNA硫基化在胞浆中相连，并共享相同的硫运输途径蛋白，由NFS1、MOCS3和可能的TUM1组成。为了剖析线粒体和胞质tRNA硫化和MOCO生物合成系统中复杂的硫转移途径，一个目标是分析新发现的两种TUM1亚型在线粒体中形成tm5s2U的作用以及在胞浆中形成mcm5s2U34和moco的作用。其目的是详细剖析硫转移途径的特殊性，并分析胞质tRNA硫化和MoCO生物合成是否存在不同的硫传递系统。此外，分析酵母和人类MOCS3之间是否存在差异是有意义的。构建的CRISPR/Cas9基因敲除细胞系(MOCS3、TUM1)的使用以及与酵母Uba4蛋白的功能互补、不同的TUM1亚型或MOCS3片段及其变体将详细揭示每种蛋白的细胞作用。总之，我们的研究将详细剖析tRNA硫基化的作用及其与人类细胞中MOCO生物合成的联系。

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.