Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae

Abstract

tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the beta-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the beta-importin family. The beta-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5.

Figure 1.

Los1 and Msn5 have different roles in tRNA nuclear export. (A) Northern analysis of pre-tRNA^Ile and mature tRNA^Ile in wild-type and mutant cells. (B) Northern analysis of pre-tRNA^Tyr and mature tRNA^Tyr in wild-type and mutant cells.

Figure 2.

FISH analysis of the role of Msn5 in tRNA nuclear-cytoplasmic dynamics. (A) Locations of tRNA^His and tRNA^Tyr in wild-type (1 and 2), los1Δ (3 and 4), and msn5Δ (5 and 6) cells. 1–6, FISH signal; 1′–6′, same cells stained with DAPI. (B) FISH analysis of the location of tRNA^Tyr in wild-type (1 and 2), msn5Δ (3 and 4), and mtr10Δ msn5Δ (5 and 6) cells in nutrient-rich (1, 3, and 5) and amino acid–deprived (2, 4, and 6) conditions. 1–6, FISH signal; 1′–6′, same cells stained with DAPI. White bar, 5 μm.

Figure 3.

Los1 and Msn5 function in the tRNA reexport process. FISH analysis for tRNA^Tyr (A) and tRNA^His (B) in wild-type fed (panels 1) or amino acid deprived (panels 2), los1Δ (panels 3 and 4), mtr10Δ (panels 5 and 6), and los1Δ mtr10Δ (panels 7 and 8) cells. 1–8, location of the tRNAs; 1′–8′, the same cells stained with DAPI. White bar, 5 μm

Figure 4.

tRNA nuclear import is constitutive. (A) Strategy to follow the subcellular dynamics of tRNA^Phe in fed and amino acid–deprived cells. Cells are drawn as circles with the cytoplasm colored light gray, and the nucleoplasm uncolored. The green balls represent the version of Trm7 [NLS-(73-151)-Trm7-GFP], which has been epitope-tagged and modified so that it is located at the INM. Trm7 specifies the tRNA 2′-O-methylcytosine modification at position 32 (Cm₃₂) indicated by the red ball on the tRNA cartoons. If tRNA^Phe enters the nucleus only when cells are deprived of amino acids, then tRNA^Phe will not bear Cm₃₂ when cells are propagated in medium with all amino acids; if import is constitutive, then tRNA^Phe will be modified in cells propagated in the presence and the absence of amino acids. (B) NLS-(73-151)-Trm7-GFP is located at the INM in wild-type and trm7Δ cells. Cells contain a plasmid encoding galactose-inducible NLS-(73-151)-Trm7-GFP and a plasmid encoding constitutively expressed nucleoporin, Nup49-mCherry, to mark the nuclear rim. NLS-(73-151)-Trm7-GFP is located as one or a few spots on the INM in both trm7Δ and wild-type cells when all amino acids are supplied or when the cells have been deprived for amino acids. White bar, 5 μm. (C) NLS-(73-151)-Trm7-GFP is stably maintained in the nucleus. Heterokaryon analysis was used to determine whether NLS-(73-151)-Trm7-GFP is maintained in the nucleus. Top row, NLS-Cca1-GFP can move from one nucleus to another in heterokaryons; middle row, H2B-GFP does not shuttle out of the nucleus that encodes this protein; bottom row, NLS-(73-151)-Trm7-GFP, like H2B-GFP, is located in only one of the nuclei of heterokaryons. White bar, 5 μm. (D) Representative HPLC chromatographs showing the quantities of m⁵C and Cm₃₂ in fed (1) and amino acid–deprived (1′) wild-type cells + vector, fed, and starved trm7Δ cells + vector (2 and 2′, respectively), and fed (3) and amino acid–deprived (3′) trm7Δ cells harboring the plasmid encoding NLS-(I73-151)-Trm7-GFP.

Figure 5.

Interactions between Los1 or Msn5 and Tef1. (A) Synthetic growth defects in los1Δ tef1Δ and msn5Δ tef1Δ double mutants. Serial dilutions were made of the indicated yeast cultures. Aliquots (5 μl) of each dilution were spotted onto solid growth medium and incubated 2-3 d at various indicated temperatures. (B) Confocal images (0.4-μm sections) of the subcellular distribution of endogenously expressed Tef1-GFP in wild-type and mutant fed and nutrient-deprived live cells. Wild-type (panels 1 and 2), los1Δ (panels 3 and 4), or msn5Δ (panels 5 and 6) mutant cells containing the constitutively expressed plasmid-encoded Nup49-mCherry were grown in SC medium lacking leucine to maintain the plasmid. Cells in 1-1″, 3-3″, and 5-5″ were further incubated in medium with all required nutrients, whereas cells in 2-2″, 4-4″, and 6-6″ were deprived of amino acids for 1.5 h. 1–6, location of Tef1-GFP; 1′–6′, location of Nup49-mCherry, demarking the nuclei rims, 1″–6″, overlay of 1–6 and 1′–6′, respectively. White bar, 5 μm. (C) Pixel intensity profiles of the subcellular distribution of Tef1-GFP in wild-type and mutant fed and nutrient deprived live cells. Pixel intensity profiles of 0.4-μm sections are shown for three independent cells for each strain and condition. The cells in B that were scanned and plotted in C are indicated with the same shape arrows or arrowheads. Red lines and axes indicate Nup49-mCherry image intensities; green lines and axes indicate Tef1–GFP intensities.