Thio-modification of Yeast Cytosolic tRNA Requires a Ubiquitin-related System That Resembles Bacterial Sulfur Transfer Systems

Abstract

The wobble uridine in yeast cytosolic tRNA Lys2 UUU and tRNA Glu3UUC undergoes a thio-modification at the second position (s 2 modification) and a methoxycarbonylmethyl modification at the fifth position (mcm 5 modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s 2 modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitinlike protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s 2 modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s 2 modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s 2 and mcm 5 modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s 2 modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.Many modified nucleotides are found in tRNAs of various organisms, and the post-translational modification of tRNA molecules is thought to be necessary to maintain their structure and thereby to exert their proper function in translation (1). In yeast, uridine of the first position of anticodon, in cytosolic tRNA (cy-tRNA), 2 for lysine (cy-tRNA Lys2 UUU ) and glutamate (cy-tRNA Glu3 UUC ) contains sulfur instead of oxygen at the second position (the s 2 modification) and 5-methoxylcarbonylmethyl at the fifth position (the mcm 5 modification). These cytRNAs read split codon boxes; they decode the general NAAtype codon and can wobble into the NAG codon (2). Thus, the s 2 and mcm 5 modifications in U 34 are thought to be important in maintaining stable codon-anticodon pairing during decoding of these cy-tRNAs on the ribosome.For the s 2 modification of cy-tRNA Lys2 UUU and cytRNA Glu3 UUC , the cysteine desulfurase Nfs1 located in the mitochondria was essential, indicating that a sulfur atom used for the s 2 modification should originate from the cysteine sulfur atom located inside the mitochondria (3). Nfs1 is also known to provide sulfur for the iron-sulfur (Fe/S) cluster biosynthesis, which involves the mitochondrial ISC and cytosolic CIA machineries (4 -6). We previously demonstrated that the s 2 modification of cy-tRNAs was dependent not only on Nfs1 but also on other ISC and CIA proteins such as Cfd1 (7). Because the cytosolic Fe/S cluster assembly mediated by CIA must precede Fe/S cluster biosynthesis in the mitochondria, which is mediated by ISC (8), our previous observation suggests that at least one cytosolic Fe/S cluster-containing protein plays an indispensable role in the s 2 modification of cy-tRNAs (7). It may also be possible that the sulfur atom forming an Fe/S cluster may itself be directly used for the s 2 modification. Besides the mitochondrial Nfs1 and Fe/S cluster assembly...

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

“…Thiocarboxylated Urm1 functions as a sulfur donor in tRNA thiolation (9)(10)(11)(12)(13). The consequences of thiocarboxylation on the ability of Urm1 to form protein conjugates have not been explored until now.…”

“…MoaD and ThiS are thiocarboxylated at their C terminus and serve as sulfur donors in molybdopterin and thiamine synthesis, respectively (8). Urm1 is also thiocarboxylated and functions as a sulfur donor in tRNA thiolation in Saccharomyces cerevisiae and mammalian cells, thus resembling prokaryotic sulfur carriers (9)(10)(11)(12)(13).…”

“…Next, MOCS3 adenylates the C terminus of Urm1, followed by the transfer of sulfur to the terminal glycine of Urm1 (15). Urm1 associates with the thiouridylases ATPBD3 (also known as CTU1 in Homo sapiens and Ncs6p in S. cerevisiae) and CTU2 (Ncs2p in S. cerevisiae), which mediate the thiolation of wobble uridines in tRNA Lys(UUU) , tRNA Gln(UUG) , and tRNA Glu(UUC) (9)(10)(11)(12)(13)(16)(17)(18).…”

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

“…MOCS3 was initially identified to be involved in molybdenum cofactor (Moco) biosynthesis in the cytosol (27). In this case, MOCS3 interacts with MOCS2A and forms a thiocarboxylate group at the C terminus of MOCS2A (24,25,27). MOCS2A subsequently assembles with MOCS2B to form the molybdopterin (MPT) synthase (28).…”

Urm1 couples sulfur transfer to ubiquitin-like protein function in oxidative stress

“…Thus, these observations suggest that Urm1 activation is more similar to that of a prokaryotic sulfur carrier protein than that of a UBL. This function has been linked to the downstream thiolation of certain tRNAs during oxidative stress, where their modification alters their decoding specificity (16)(17)(18)(19).…”