Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae

Abstract

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082-2085]. Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export. We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast. Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs. Saturation of tRNA export pathway is a possible explanation of this phenomenon.

Figure 1

Nuclear accumulation of tRNAs in nup116Δ cells. Parent W303 (A–D) and nup116Δ strain SWY27 (E–H) were grown at 23°C, and log phase cells were shifted to 37°C for 2 h. FISH was used to detect poly(A) RNA (A and E), tRNA^Ile (B and F), tRNA^Met (C and G), and tRNA^Tyr (D and H). a–h are 4′,6-diamidino-2-phenylindole staining of cells shown in A–H, respectively. (Bar = 10 μm.)

Figure 2

Aminoacylation of steady-state tRNA^Met. tRNAs were isolated from W303 at pH 9 (lane 1) and at pH 4.5 (lane 2); SWY27, nup116Δ (lane 3); A364A (lane 4); ils1-1 (lane 5); SS328 (lane 6); and mes1-1 (lane 7). Acid gel Northern analysis was performed. Arrows indicate aminoacylated tRNAs, and arrowheads indicate nonaminoacylated tRNAs.

Figure 3

Aminoacylation status of newly synthesized tRNA. Cells were incubated at 37°C for either 10 min (lanes 1–8) or 1 h (lanes 9–12) before addition of [³H]uracil. Aminoacylated (lanes 1, 3, 5, 7, 9, and 11) and nonaminoacylated (lanes 2, 4, 6, 8, 10, and 12) tRNAs were isolated from W303 wild-type cells (lanes 1, 2, 9, and 10); SWY27, nup116Δ (lanes 3, 4, 11, and 12); NT33-5/YCpcca-M1, wild-type (lanes 5 and 6); and NT33-5/YCp50, cca1-1 (lanes 7 and 8; ref. 19) in the absence (lanes 1–8) or presence (lanes 9–12) of a 10-fold excess of nonaminoacylated yeast tRNA. RNAs were resolved on an acidic polyacrylamide gel and detected by fluorography.

Figure 4

Defect in tRNA nuclear export in aminoacyl-tRNA synthetase mutants. A364A (A–D), mes1-1 (E–H), ils1-1 (I–L), and tys1-1 (M–P) were grown at 23°C, and log phase cells were shifted to 37°C for 2 h. FISH was used to detect poly(A) RNA (A, E, I, and M), tRNA^Ile (B, F, J, and N), tRNA^Met (C, G, K, and O), and tRNA^Tyr (D, H, L, and P). Arrows show locations of nuclei. Arrowheads indicate signal from nucleus-located intron-containing pre-tRNA^Tyr. (Bar = 10 μm.)

Figure 5

(Upper) Restriction map of TYS1 and flanking region. Numbers 1, 2, and 3 are reading frames from one direction, and −1, −2, and −3 are reading frames from the opposite direction. The half bars represent ATG codons, and the full bars represent stop codons. A segment of UBR1, which codes for ubiquitin-protein ligase, is present in the DNA fragment. TYS1 encodes tyrosyl-tRNA synthetase. TFG1 encodes the large subunit of transcription factor TFIIF. HGH1 encodes HMG1/2 homologue. BUB1 encodes a serine/threonine protein kinase. Abbreviations for restriction enzymes are as follows: B, BamHI; S, SacI; Sa, SalI. (Lower) Mapping of the TYS1 gene. Complete rescue of the growth of tys1-1 at 37°C is shown by + and nonrescue by − for each fragment.