Thio modification of yeast cytosolic tRNA is an iron-sulfur protein-dependent pathway

Abstract

Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.

FIG. 1.

Cfd1 is required for 2-thio modification of cy-tRNAs but not of mt-tRNAs. (A) Total tRNAs prepared from TH-CFD1 cells grown without (−) or with (+) doxycycline (Dox) for 24 h (left panels) and from Gal-NFS1/YN cells grown with galactose for 20 h (Gal) or glycerol for 48 h (Gly) (right panels) were used. Isolated tRNAs (0.05 OD₂₆₀ unit [2 μg]) were loaded onto APM-containing (top panels) and APM-lacking (middle panels) denaturing gels, and after electrophoresis they were blotted to nylon membranes. These blots were hybridized with oligonucleotide probes specific to cy-tRNA_UUU^Lys2(cyK), cy-tRNA_UUC^Glu3(cyE), mt-tRNA_UCU^Arg(cyR), mt-tRNA_UUU^Lys(mtK), and mt-tRNA_UUG^Gln(mtQ). Positions of 2-thio-modified tRNAs that exhibited mobility retardation are indicated by a bracket, and positions of non-thio-modified tRNAs are indicated by an arrowhead. The protein levels of Cfd1 and Nfs1 were examined at time points similar to those described above by immunoblotting (bottom panels). (B) The relative proportions of 2-thio-modified tRNA in each tRNA were estimated. After the APM-containing gel electrophoresis shown in panel A, radioactivity bound to either the 2-thio-modified form or the unmodified form was measured as described in Materials and Methods. Values are represented as percentages of the amount of 2-thio-modified tRNA and the total amount of each tRNA (i.e., both the 2-thio modified and unmodified tRNA).

FIG. 2.

Unmodified cy-tRNA_UUU^Lys2accumulated in Cfd1-depleted cells can be thio modified by Cfd1-replete cytosol in vitro. Total tRNAs prepared from Cfd1-depleted cells (lane 1) were examined for 2-thio modification by the addition of yeast cytosolic fraction prepared from wild-type cells (lane 3). A 10-fold-higher amount of the cytosolic fraction of the wild-type cells is shown in lane 4 (+++). Total tRNAs prepared from the wild-type cells (lane 2) were also analyzed as controls. The 2-thio-modified and unmodified cy-tRNA_UUU^Lys2was detected and shown as in Fig. 1.

FIG. 3.

Both Nbp35 and Cia1 are required for the 2-thio modification of cy-tRNA_UUU^Lys2, while the essential cytosolic Fe/S protein Rli1 is not. (A) The 2-thio modifications in Gal-NBP35/YN cells grown with galactose for 16 h (Gal) or with glycerol for 60 h (Gly) were examined by APM-Northern analysis with specific probes against cy-tRNA_UUU^Lys2(cyK) and mt-tRNA_UUU^Lys(mtK). An oligonucleotide probe against mt-tRNA_UCU^Arg(cyR) was also used. The bottom panel represents the protein level of Nbp35 in the corresponding cells analyzed by immunological methods. The relative proportions of 2-thio-modified tRNAs are depicted in panel B as described in the legend to Fig. 1B. (C) The 2-thio modifications of cy-tRNA_UUU^Lys2in Gal-CIA1 cells grown with galactose for 16 h (Gal) and with glycerol for 60 h (Gly) and in TH-RLI1 cells grown without doxycycline (Dox) for 23 h (−) and with Dox for 50 h (+) were monitored as in panel A. The protein levels of Cia1 and Rli1 (bottom panels) and the relative proportions of the 2-thio-modified tRNAs (D) are also shown.

FIG. 4.

Extramitochondrial Nfs1 is not sufficient for 2-thio modification of cy-tRNA_UUU^Lys2. Gal-NFS1/YN cells without plasmid-borne Nfs1 (lane 1), with plasmid-borne Nfs1 lacking mitochondrial presequence (lane 2), or with plasmid-borne full-length Nfs1 precursor to be localized in the mitochondria (lane 3) were grown with glucose for 50 h, and their total tRNAs were subjected to APM-Northern analysis with cy-tRNA_UUU^Lys2-specific oligonucleotide probe (cyK) and with mt-tRNA_UUU^Lys-specific oligonucleotide probe (mtK). The 2-thio-modified and unmodified tRNAs are indicated as in Fig. 1. The bottom panel shows the levels of Nfs1 analyzed by immunological detection.

FIG. 5.

Depletion of the essential mitochondrial Isu scaffold proteins causes impairment in the 2-thio modification of cy-tRNA_UUU^Lys2but not of mt-tRNA_UUU^Lys. (A) Isu1 levels were determined by immunological analysis with anti-Isu1 antibody in Gal-ISU1/Δisu2 cells grown with galactose (Gal) or glycerol (Gly) for 48 h. (B) Total tRNAs of Gal-ISU1/Δisu2 cells grown with galactose (Gal) for 12 h or glycerol (Gly) for 49 h and 72 h were examined by APM-Northern analysis. Total tRNAs of Gal-NFS1/YN cells were tested in a similar fashion. The 2-thio modifications of cy-tRNA_UUU^Lys2(left) andmt-tRNA_UUU^Lys(right) were analyzed by the APM-Northern method. The 2-thio-modified and unmodified tRNAs are shown as in Fig. 1C. (C) The 2-thio modifications of cy-tRNA_UUU^Lys2and mt-tRNA_UUU^Lyswere examined with the single deletion mutants Δisu1 and Δisu2. These cells, both of which showed normal growth rates, were grown with glucose for 20 h. Gal-ISU1/Δisu2 cells grown with glucose medium for 48 h to repress the expression of Isu1 in the absence of Isu2 were analyzed in parallel. The same amounts of tRNA samples were examined in a denaturing gel with APM (upper left panel) and without APM (w/o APM). The oligonucleotide probes used were the same as shown in Fig. 1. The right panel indicates the relative proportions of 2-thio-modified tRNAs estimated from APM-Northern analysis shown in the upper left panel, as described in the legend to Fig. 1B.