maf1Δ cells accumulate tRNA precursors. maf1Δ and wild type (wt) control strain BY4742 were grown to mid-log phase in rich glucose medium (YPD), and one-half of each culture was shifted to rich medium containing glycerol (YPGly) and cultivated for 2 h at 37 °C. A–C, Northern analysis of intron-containing tRNA^Phe(GAA) and tRNA^Leu(CAA) using probes that recognize both pre-tRNA and mature tRNA (A); intron-containing tRNA^Leu(CAA) using distinct probes, specific to precursors or mature tRNA (B); and intron-less tRNA_i^Met(CAU) and tRNA^His(GUG) (C). The sequences of tRNA-specific oligonucleotide probes are specified under “Experimental Procedures.” The positions of initial transcripts, end-processed intron-containing pre-tRNA, and mature tRNA are indicated. Pre-tRNAs and mature tRNAs were quantified. The bars represent levels for primary transcript retaining 5′ and 3′ termini, intron-containing precursor, and mature form normalized to loading control. The fold of change in maf1Δ cells cultivated under indicated conditions was calculated relative to expression in control strain (wt) at standard conditions (YPD). Standard deviation was calculated on the basis of three independent experiments.

pre-tRNA levels depend on nuclear Maf1. A, wild type BY4742 (wt), maf1Δ, msn5Δ, and maf1Δ msn5Δ cells were grown to mid-log phase in rich glucose medium (YPD), and one-half of each culture was shifted to rich medium containing glycerol (YPGly) and cultivated for 2 h at 37 °C. Northern hybridization was performed with probes specific to pre-tRNA^Leu and mature tRNA^Leu(CAA). 5.8 S rRNA served as a loading control. B, quantification of primary transcripts (–▭–▭–) and end-matured, intron-containing pre-tRNA^Leu(CAA) (▭–▭) levels. The fold of change in each strain cultivated under indicated conditions was calculated relative to expression in control strain (wild type) at the same growth conditions. Standard deviation was calculated on the basis of three independent experiments.

Transcription inhibition prevents accumulation of pre-tRNA in maf1Δ cells. maf1Δ and wild type cells were grown to mid-log phase in rich glucose medium (YPD) and then were treated with 5 μg/ml thiolutin. The cells were harvested just before (time 0) and after thiolutin treatment at the indicated time points. A, Northern analysis was performed with probe recognizing pre-tRNA^Leu(CAA). The positions of primary transcript and end-matured, intron-containing precursor were indicated. U3 snoRNA served as a loading control. wt, wild type. B, quantification of pre-tRNA^Leu(CAA) levels in wild type (solid lines and closed squares) and maf1Δ (dashed lines and open squares). The curves show the ratio of primary transcripts (–▭–▭–) and end-matured, intron-containing pre-tRNA^Leu(CAA) (▭–▭) at each time point relative to its levels in the wild type strain immediately before thiolutin addition (time 0). Each value itself was first normalized to U3 snoRNA. Standard deviation was calculated on the basis of three independent experiments.

Overexpression of RPR1 gene has no effect on pre-tRNA accumulation in maf1Δ cells. Wild type BY4742 (wt) and maf1Δ cells transformed with control plasmid pFL44L (designated []) or overexpressing RPR1 ([RPR1]) were grown in SC-ura medium containing 2% glucose, shifted to SC-ura medium containing 2% glycerol, and cultivated for 2 h at 37 °C. A, Northern hybridization was performed with probes specific to RNA RPR1, pre-tRNA^Leu(CAA), or U3 snoRNA (used as a control). B, bars indicate the ratio of initial transcript and end-matured, intron-containing pre-tRNA^Leu(CAA) levels relative to their levels in wild type cells bearing control plasmid pFL44L (designated wt/[]). The levels of RPR1 precursor and its mature form were quantified. The bars represent the ratio of both RPR1 transcripts relative to their levels in wild type cells bearing control plasmid pFL44L (designated wt/[]). Each value itself was first normalized to U3 snoRNA. Standard deviation was calculated on the basis of three independent experiments.

Cytoplasmic splicing endonuclease is not limiting for processing of intron-containing pre-tRNA that accumulate in maf1Δ cells. Wild type W303 (wt), W303 maf1Δ (maf1Δ), or control W303 sen2-24 (sen2-24) strains were transformed with plasmids bearing cytoplasmic tRNA splicing endonuclease genes under control of galactose-inducible promoter. As a control, we used the same strains transformed with the empty vectors. Transformants were grown in minimal glucose medium supplemented as required. + indicates induction by galactose addition to final concentration 2% for 2 h; − indicates no induction; 23 and 37 °C are the temperature conditions. Northern hybridization was performed with probes specific to tRNA^Ile(UAU). Lane 1, maf1Δ with induced plasmids bearing cytoplasmic tRNA splicing endonuclease genes; lane 2, maf1Δ with induced empty vectors; lane 3, maf1Δ with uninduced plasmids bearing cytoplasmic tRNA splicing endonuclease genes; lane 4, maf1Δ with uninduced empty vectors; lane 5, wild type with induced plasmids bearing cytoplasmic tRNA splicing endonuclease genes; lane 6, wild type with induced empty vectors; lane 7, wild type with uninduced plasmids bearing cytoplasmic tRNA splicing endonuclease genes; lane 8, wild type with uninduced empty vectors; lane 9, sen2-42 with uninduced plasmids bearing cytoplasmic tRNA splicing endonuclease genes at 23 °C; lane 10, sen2-42 with uninduced plasmids bearing cytoplasmic tRNA splicing endonucleases genes at 37 °C; lane 11, sen2-42 with induced plasmids bearing cytoplasmic tRNA splicing endonuclease genes at 23 °C; lane 12, sen2-42 with induced plasmids bearing cytoplasmic tRNA splicing endonuclease genes at 37 °C. RNAs for lanes 9–12 were extracted and probed at the same times as those in lanes 1–8 but were run on a separate gel.

maf1Δ cells accumulate pre-tRNA^Leu(CAA) in the nucleus. maf1Δ and control wild type cells were grown in glucose medium (YPD) at 30 °C to early log phase. Part of the culture was shifted to glycerol medium (YPGly) for 2 h at 37 °C. The cultures were harvested and subjected to fluorescence in situ hybridization. The Cy3-labeled probe specific to sequence of pre-tRNA^Leu(CAA) intron and DAPI staining were applied.

Los1 is limiting for intron-containing tRNA export and processing in maf1Δ cells. Wild type (wt) BY4742, los1Δ, and maf1Δ cells, transformed with control plasmid Yep24 (designated []) or overexpressing LOS1 ([LOS1]) were grown in SC-ura glucose medium. Part of the culture was shifted to minimal glycerol medium (Sc-ura + glycerol) for 2 h at 37 °C. A, Northern hybridization was performed with probes specific to pre-tRNA^Leu(CAA) or U3 snoRNA (used as a control). B, quantification of the pre-tRNA^Leu(CAA) levels. The bars represent levels for primary transcript retaining 5′ and 3′ termini and intron-containing precursor of tRNA^Leu(CAA) normalized to U3 snoRNA transcript. The fold of change in each strain was calculated relative to expression in wild type strain bearing control plasmid YEp24 (designated as wt/[]). The standard deviation was calculated on the basis of three independent experiments.

Localization of Los1 exportin is affected by growth conditions but not by Maf1. Strains BY4742 LOS1-Myc (wt) and BY4742 maf1Δ LOS1-Myc (maf1Δ) were grown to early logarithmic phase in YPD. Part of the culture was shifted to glycerol medium (YPGly) for 2 h at 37 °C. Localization of Los1 was examined by indirect immunofluorescence microscopy using anti-Myc antibodies. Nuclear DNA was stained with DAPI.

Gcn4 induction in maf1Δ cells. A GCN4-lacZ (CEN, URA) reporter plasmid was introduced into wild type and maf1Δ mutant cells. Transformants were grown in YPD grown to early log phase in rich glucose medium (YPD) at 30 °C, then transferred to glycerol medium (YPGly), and incubated at 37 °C for 2 h. A, β-galactosidase activity was measured in yeast extracts and correlated to protein concentration (arbitrary units). B, total proteins were isolated as described under “Experimental Procedures.” Equal amounts of proteins were resolved on 10% SDS-polyacrylamide gel and subjected to immunoblot analysis using specific rabbit anti-Gcn4 or mouse anti-Act1 antibodies (used as a loading control).

Maf1-mediated transcription control and nuclear export by Los1 are regulated by environmental conditions in coordinated manner. A, under favorable growth conditions in glucose medium Maf1 is phosphorylated, thus preventing Pol III repression, and Los1 is localized in nuclear membrane providing active export of intron-containing tRNA precursors (wt panel). tRNA synthesis is increased in the absence of Maf1, but export is saturated (maf1Δ panel). B, under repressive conditions on glycerol medium, both tRNA synthesis and tRNA export are decreased, respectively, by Maf1-Pol III interaction and Los1 redistribution to the cytoplasm (wt panel). This coupling is lost in the absence of Maf1, resulting in nuclear accumulation of intron containing pre-tRNAs (maf1Δ panel).