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. 2008 Nov 25;105(47):18255-60.
doi: 10.1073/pnas.0808756105. Epub 2008 Nov 18.

A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway

Christian D Schlieker  1 , Annemarthe G Van der Veen, Jadyn R Damon, Eric Spooner, Hidde L Ploegh
Affiliations

A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway

Christian D Schlieker et al. Proc Natl Acad Sci U S A. .

Abstract

Urm1 is a highly conserved ubiquitin-related modifier of unknown function. A reduction of cellular Urm1 levels causes severe cytokinesis defects in HeLa cells, resulting in the accumulation of enlarged multinucleated cells. To understand the underlying mechanism, we applied a functional proteomics approach and discovered an enzymatic activity that links Urm1 to a tRNA modification pathway. Unlike ubiquitin (Ub) and many Ub-like modifiers, which are commonly conjugated to proteinaceous targets, Urm1 is activated by an unusual mechanism to yield a thiocarboxylate intermediate that serves as sulfur donor in tRNA thiolation reactions. This mechanism is reminiscent of that used by prokaryotic sulfur carriers and thus defines the evolutionary link between ancient Ub progenitors and the eukaryotic Ub/Ub-like modification systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Depletion of Urm1 results in a cytokinesis defect. (A) Efficiency of Urm1 knockdown in HeLa cells 4 days after lentiviral transduction of luciferase (control) or Urm1 shRNA constructs. (B) Confocal microscopy of Urm1-depleted HeLa cells. Red, Hoechst; green, actin. (Scale bars, 10 μm.) (C) Progress through cell cycle was assessed by quantification of DNA content in wild-type, shGFP, and shUrm1 HeLa cells. The DNA content of cells was quantified by flow cytometry 12 h after release from a double-thymidine block and plotted in a histogram.
Fig. 2.
Fig. 2.
A Urm1-based functional probe reacts with ATPBD3, a Urm1-directed enzymatic activity. (A) HA-Urm1-VME reacts with ATPBD3 to yield a covalent adduct. Radiolabeled ATPBD3 was synthesized by in vitro translation, reacted with HA-Urm1-VME in the absence or presence of NEM, and resolved by SDS/PAGE. (B) HA-Urm1-VME retrieves ATPBD3 from cell lysates. HEK293T cells were transfected with FLAG-ATPBD3 or control vector (−), lysed, exposed to HA-Urm-VME, and immunoprecipitated (IP) with anti-HA antibodies. Anti-FLAG antibodies were used for detection. (C) Overexpressed ATPBD3 associates with endogenous UPF0432 and Urm1 in vivo. HEK293T cells were transfected with ATPBD3-HA, lysed, and immunoprecipitated with anti-HA antibodies. After SDS/PAGE and silver staining, the indicated ATPBD3-associated proteins were identified by MS.
Fig. 3.
Fig. 3.
Urm1 is charged with sulfur in vivo to form of a C-terminal thiocarboxylate. (A) HEK293T cells were cotransfected with MOCS3-HA and HA-Urm1, lysed, and subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitates were resolved by SDS/PAGE and subjected to silver staining. Note that both proteins were HA-tagged, i.e., the appearance of MOCS3-HA and HA-Urm1 is not indicative of a mutual interaction. (B) The band marked in A was excised, digested with Asp-N, and analyzed by LC-MS/MS. The observed parent ion masses are consistent with Asp-N-released peptides from the C terminus of Urm1 and suggest that both unmodified and thiocarboxylated Urm1 were present (the mass difference between sulfur and oxygen is 16 Da). (C) Note that the y-ion series (i.e., the series containing the C terminus), derived from fragmentation of the smaller parent ions depicted in B, is in excellent agreement with the location of the sulfur at the C terminus. Masses in brackets are theoretical and were not observed in this experiment.
Fig. 4.
Fig. 4.
Urm1 is required for the thiolation of cytosolic tRNAs. (A) U-rich anticodons of several cytosolic tRNAs, exemplified by the human tRNALYS(UUU), contain a modified uracil, mcm5S2U, at position 34. (B) Urm1 and Urm1-associated enzymatic activities are required for tRNA thiolation in S. cerevisiae. RNA was isolated from the indicated S. cerevisiae strains, resolved by denaturing PAGE, and subjected to Northern blotting. Polyacrylamide gels supplemented with APM (Top) were used to discriminate between thiolated and hypomodified tRNAs, a gel devoid of APM served as control (Bottom). The indicated nonthiolated tRNAHis serves as control (Middle). Radioactive oligonucleotides with the indicated specificities served as probes. (C) Urm1 and ATPBD3 contribute to tRNA thiolation in human cells. RNA isolated from the indicated HeLa cell lines was analyzed as in B, purified unmodified tRNALys(UUU) (5 pmol) served as control. (D) Comparison of thiolation levels obtained from a densiometric quantification of the bands seen in C.
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