Beyond tRNA cleavage: novel essential function for yeast tRNA splicing endonuclease unrelated to tRNA processing

Abstract

Pre-tRNA splicing is an essential process in all eukaryotes. In yeast and vertebrates, the enzyme catalyzing intron removal from pre-tRNA is a heterotetrameric complex (splicing endonuclease [SEN] complex). Although the SEN complex is conserved, the subcellular location where pre-tRNA splicing occurs is not. In yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates, pre-tRNA splicing is nuclear. We engineered yeast to mimic the vertebrate cell biology and demonstrate that all three steps of pre-tRNA splicing, as well as tRNA nuclear export and aminoacylation, occur efficiently when the SEN complex is nuclear. However, nuclear pre-tRNA splicing fails to complement growth defects of cells with defective mitochondrial-located splicing, suggesting that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. The novel function requires all four SEN complex subunits and the catalytic core. A subset of pre-rRNAs accumulates when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing. Thus, findings suggest that selection for the subcellular distribution of the SEN complex may reside not in its canonical, but rather in a novel, activity.

Figure 1.

Subcellular location of tRNA splicing machinery in yeast. (A) Location of Tpt1-GFP and Nup49-mCherry, and merged images. (B) Nuclear redistribution of tRNA SEN subunits and tRNA ligase. Location of plasmid-encoded NLS-tagged and 2GFP-tagged tRNA SEN subunits (NLS-Sen) or tRNA ligase (NLS-Trl1) in cells harboring Nup49-mCherry. (C) Heterokaryon analyses of nuc SEN complex subunits. Yeast expressing GFP-tagged NLS-Sen, NLS-H2B, or NLS-Cca1 were mated with the kar1-1 mutant to generate heterokaryons (outlined). Cartoons of heterokaryons on the right depict either shuttling (two green nuclei) or nonshuttling (one green nucleus) results. DAPI staining shows visualization of nuclear DNA. More than 20 heterokaryons were counted, and the same results were obtained as depicted in the figure. Bar, 4 μm.

Figure 2.

Ability of nuclear-localized tRNA splicing machinery to catalyze pre-tRNA^Leu_CAA splicing. (A) Correction of splicing defect of sen2-42 by the nuc SEN complex and nuc-cyt ligase. Northern analysis of sen2-42 cells containing either vectors (V) or the set of plasmids encoding the nuc SEN complex and nuc-cyt ligase (N) in the presence (+) or absence (−) of Gal induction at the permissive temperature (23°C) or after 2 h at the np temperature (37°C). (B) Correction of tRNA ligation defects of rlg1-4 by the nuc SEN complex and nuc-cyt ligase. Northern analysis of rlg1-4 cells containing the same set of plasmids as A under the same conditions. (P) Initial pre-tRNA transcript; (i) end-matured intron-containing pre-tRNA; (m) mature tRNA; (h) half tRNAs; (5S) loading control; (Q_m) quantifications of m signal intensity. Ratios of the signal intensities of i/p are shown. (C) 2′-Phosphotransferase activity. RT–PCR was used to assess whether spliced tRNA possess a 2′-phosphate at the splice junction of tRNA^Ile. (Lane 1) tpt1Δ cells with Gal-inducible TPT1 in the presence of Gal. (Lane 2) The same cells as lane 1 without Gal induction. (Lanes 3,4) sen2-42 cells with the set of plasmids encoding nuclear-localized splicing machinery in the presence of Gal at 37°C. (Lane 5) Wild-type cells.

Figure 3.

FISH analyses of tRNA^Tyr_GUA in yeast expressing nuclear tRNA splicing machinery. (A) Wild type (wt). (B) los1Δ. (C) sen2-42 cells expressing vectors. (D) sen2-42 mutants with the plasmids encoding the nuc SEN complex and nuc-cyt ligase. Cells were assessed after growth at 23°C or after a 2-h shift to 37°C, and the location of tRNA^Tyr was determined. DAPI was used to locate nuclear DNA. Bar, 5 μm.

Figure 4.

Efficient tRNA aminoacylation in cells possessing nuclear tRNA splicing. (A) Assessment of tRNA^Leu_UAG aminoacylation by Northern analysis of tRNA from W303 (wt) Leu⁻ cells or sen2-42 cells containing either vectors (V), the set of plasmids encoding the nuc SEN complex and nuc-cyt ligase (N), or the set of plasmids encoding the mito SEN complex and cytoplasmic ligase (M) in the presence of Gal induction at the permissive temperature (23°C) or after 2 h at the np temperature (37°C). (C) Aminoacylated tRNA^Leu_UAG; (U) nonaminoacylated tRNA^Leu_UAG. (B) Assessment of tRNA aminoacylation with Gcn4 translation using a β-galactosidase reporter assay. sen2-42 cells containing either vectors (V) or the set of plasmids encoding the nuc SEN complex and nuc-cyt ligase (N) in the presence (+aa) or absence (−aa) of amino acids.

Figure 5.

The mitochondrial tRNA SEN complex and cytoplasmic tRNA ligase can rescue the ts growth phenotype of sen2-42, whereas the nuclear tRNA splicing machinery does not. Wild-type (wt) or sen2-42 (sen2) cells with the set of vectors (V) or the set of Gal-inducible plasmids encoding the nuc SEN complex and nuc-cyt ligase (N) or the mito SEN complex and cytoplasmic ligase (M). The spot assay was accomplished by placing an equal number of serially diluted cells on selective medium containing Gal (Gal) or lacking Gal (Glu) and incubated at the permissive temperature (23°C) (A,B,E,F) or the np temperature (37°C) (C,D,G,H).

Figure 6.

All four SEN subunits are required in the cytoplasm for viability. (A) Assessment of whether depletion of the endogenous mitochondrial tRNA SEN can be complemented with nuclear tRNA splicing. Yeast expressing tet-regulated tRNA SEN subunits (tet sen) were transformed with sets of plasmids encoding either the Nuc SEN complex and nuc-cyt ligase or the mito SEN complex and cyt ligase. Growth after 5 d was assessed on Gal-containing medium in which cells were depleted (+DOX) or not depleted (−DOX) of the endogenous tet-regulated tRNA SEN subunit. Where colonies are absent, no colonies appeared after >7-d incubation. (B) Assessment of whether the nuclear tRNA splicing defect of tet sen34 can be complemented by nuclear tRNA splicing. The yeast tet sen34 strain containing either vectors (V) or the set of plasmids encoding the nuc SEN complex and nuc-cyt ligase (N) were assessed for nuclear tRNA splicing by northern analysis in the presence (+) or absence (−) of either Gal or DOX. Subunits were depleted by addition of DOX for 9 h. (P) Initial pre-tRNA transcript; (i) end-matured intron-containing pre-tRNA; (m) mature tRNA; (5S) loading control.

Figure 7.

The catalytic core of Sen2 for tRNA splicing is required for the novel function of the tRNA SEN complex at cytoplasm and does not affect nuclear tRNA splicing by the nuc SEN complex. sen2-42 cells were transformed with the set of plasmids encoding either the mito SEN complex (M), vectors (V), or the Nuc SEN complex (N) in addition to the presence (+) or absence (−) of catalytically inactive sen2 (H297A). (A) Transformants were assessed for growth in a spot assay in selective medium containing Gal at the permissive temperature (23°C) or np temperature (37°C). (B) Transformants were assessed for tRNA splicing defects. (p) Precursor tRNA; (i) end-processed intron-containing precursor tRNA; (m) mature tRNA; (H) catalytically inactive sen2 H297A.

Figure 8.

Northern analyses of pre-rRNA processing in sen2-42 mutants expressing nuclear tRNA splicing machinery. sen2-42 cells were transformed with either the set of plasmids encoding the mito SEN complex and cyt ligase (M), the vectors (V), or the set of plasmids encoding the nuc SEN complex and nuc-cyt ligase (N). RNAs from cells induced with Gal for 2 h and incubated at 23°C or for 2 h at 37°C were resolved on an agarose–formaldehyde gel and transferred to a membrane, and the membrane was probed for 27SA2, 27S, and 20S pre-rRNAs and 25S and 18S rRNAs.