The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA

Abstract

The biogenesis of transfer RNA is a process that requires many different factors. In this study, we describe a genetic screen aimed to identify gene products participating in this process. By screening for mutations lethal in combination with a sup61-T47:2C allele, coding for a mutant form of, the nonessential TAN1 gene was identified. We show that the TAN1 gene product is required for formation of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA. In Saccharomyces cerevisiae, ac(4)C is present at position 12 in tRNAs specific for leucine and serine as well as in 18S ribosomal RNA. Analysis of RNA isolated from a tan1-null mutant revealed that ac(4)C was absent in tRNA, but not rRNA. Although no tRNA acetyltransferase activity by a GST-Tan1 fusion protein was detected, a gel-shift assay revealed that Tan1p binds tRNA, suggesting a direct role in synthesis of ac(4)C(12). The absence of the TAN1 gene in the sup61-T47:2C mutant caused a decreased level of mature, indicating that ac(4)C(12) and/or Tan1p is important for tRNA stability.

FIGURE 1.

Cloverleaf structure of . The position of the sup61-T47:2C mutation and modified nucleosides are indicated where numbers within the circles represent the following modifications: 1, N⁴-acetylcytidine (ac⁴C); 2, dihydrouridine (D); 3: 2′-o-methylguanosine (Gm); 4, N²,N²-dimethylguanosine (G); 5, N⁶-isopentenyladenosine (i⁶A); 6, pseudouridine (Ψ); 7, 2′-O-methyluridine (Um); 8, 5-methylcytidine (m⁵C); 9, 5-methyluridine (m⁵U); and 10, Etcheverry et al. (1979) detected a modification at position 57 or 58 that presumably corresponds to 1-methyladenosine (m¹A) at position 58.

FIGURE 2.

The TAN1 gene is required for growth of a sup61-T47:2C strain. (A) Wild-type (UMY2220), sup61-T47:2C (UMY2256), tan1Δ (UMY2874), and sup61-T47:2C tan1Δ (derived from UMY2951) strains carrying the low copy URA3 plasmid pRS316-TAN1 were grown in synthetic complete medium (SC), serially diluted, spotted onto SC and SC+5-fluoro-orotic acid (5-FOA) plates, and incubated at 30°C for 2 d. Cells containing a URA3 plasmid are unable to grow on 5-FOA–containing media (Boeke et al. 1984). (B) The sup61-T47:2C tan1Δ strain in A transformed with pRS425 or pRS425-sup16⁺ (high copy LEU2 plasmids) was grown in SC-Leu medium, serially diluted, spotted onto SC-Leu and SC-Leu+5-FOA plates, and incubated for 3 d at 30°C.

FIGURE 3.

The tan1-null mutant lacks ac⁴C in tRNA. HPLC analysis of nucleosides derived from total tRNA isolated from wild-type strain (UMY2220) carrying pRS316 (A); wild-type strain (UMY2220) carrying pRS316 with addition of 0.5 nmole synthetic ac⁴C (B); tan1Δ strain (UMY2874) carrying pRS316 (C); and tan1Δ strain (UMY2874) carrying pRS316-TAN1 (D). Only the portion of the chromatograms between retention times 30 and 35 min are shown. An arrow indicates the peak corresponding to ac⁴C. (E) UV absorption spectrum of the peak indicated in A. (F) UV absorption spectrum of synthetic ac⁴C.

FIGURE 4.

The Tan1 protein interacts with tRNA. (A) SDS-PAGE analysis of GST-Tan1 protein purified from E. coli. The gel was stained with Coomassie brilliant Blue R-250. (Lane 1) Molecular weight standard (BenchMark protein ladder, Invitrogen). (Lane 2) E. coli crude extract with expressed GST-Tan1 protein. (Lane 3) Purified GST-Tan1 protein. (B) Gel mobility shift assay using ³²P-labeled T7-transcribed and increasing amounts (0.15, 0.3, and 0.6 μg) of GST-Tan1 protein. The supershift was induced by monoclonal anti-GST antibody. (C) Gel mobility shift assay using ³²P-labeled T7-transcribed and increasing amounts (0.15, 0.3, and 0.6 μg) of GST-Tan1 protein.

FIGURE 5.

The TAN1 gene product stabilizes the mutant form of . Northern blot analysis of total RNA isolated from wild-type (UMY2220), sup61-T47:2C (UMY2256), tan1Δ (UMY2874), and sup61-T47:2C tan1Δ (derived from UMY2951) strains, carrying pRS425-sup16⁺, grown in SC-Leu medium at 30°C. The blot was probed simultaneously for pre-, , and (see Materials and Methods). The position and identity of (black) and (gray) species are indicated on the right.