In vivo biochemical analyses reveal distinct roles of β-importins and eEF1A in tRNA subcellular traffic

Abstract

Bidirectional tRNA movement between the nucleus and the cytoplasm serves multiple biological functions. To gain a biochemical understanding of the mechanisms for tRNA subcellular dynamics, we developed in vivo β-importin complex coimmunoprecipitation (co-IP) assays using budding yeast. Our studies provide the first in vivo biochemical evidence that two β-importin family members, Los1 (exportin-t) and Msn5 (exportin-5), serve overlapping but distinct roles in tRNA nuclear export. Los1 assembles complexes with RanGTP and tRNA. Both intron-containing pre-tRNAs and spliced tRNAs, regardless of whether they are aminoacylated, assemble into Los1-RanGTP complexes, documenting that Los1 participates in both primary nuclear export and re-export of tRNAs to the cytoplasm. In contrast, β-importin Msn5 preferentially assembles with RanGTP and spliced, aminoacylated tRNAs, documenting its role in tRNA nuclear re-export. Tef1/2 (the yeast form of translation elongation factor 1α [eEF1A]) aids the specificity of Msn5 for aminoacylated tRNAs to form a quaternary complex consisting of Msn5, RanGTP, aminoacylated tRNA, and Tef1/2. Assembly and/or stability of this quaternary complex requires Tef1/2, thereby facilitating efficient re-export of aminoacylated tRNAs to the cytoplasm.

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA

“…Intriguingly, the top ranked "cellular component" result indicated enrichment for translational elongation complex 1 (Table 3), a tetrameric GTP exchange complex that mediates enzymatic delivery of aminoacyl tRNAs to the A-site of the ribosome during translation (Negrutskii and El'skaya 1998; see also Huang and Hopper 2015).…”

Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking

“…The Mtr10 ␤-importin and the Dhh1 and Pat1 P-body machinery components have been implicated in tRNA nuclear import, as cells with mutations in these genes are unable to accumulate cytoplasmic tRNAs in the nucleus; however, there is no evidence that these proteins directly move tRNA from the cytoplasm to the nucleus (31,58,63,73). Yeast cells harboring mtr10⌬ or dhh1⌬ pat1⌬ mutations possess elevated levels of both end-extended and m 2 2 G hypomodified spliced tRNAs, supporting the idea of a role for tRNA retrograde nuclear import in tRNA quality control for end maturation and m 2 2 G modification (72).…”

“…In contrast, Msn5 preferentially exports mature aminoacylated tRNAs in the tRNA reexport step (see below). To specifically and efficiently export aminoacylated tRNA to the cytoplasm, Msn5 forms a quaternary complex with nuclear pools of translation elongation factor 1␣ (eEF1A), RanGTP, and aminoacylated tRNA (58). Since such aminoacylated tRNAs have first been proofread by the nuclear pools of aminoacyl-tRNA synthetases, Msn5 also contributes to tRNA quality control.…”

“…1A). Indeed, recent in vivo analyses of Los1-tRNA-RanGTP nuclear export complexes demonstrated that Los1 can form export complexes with both charged tRNA and uncharged tRNA (58).…”

Molecular mechanisms regulating plant infection in the rice blast fungus Magnaporthe oryzae

“…Mns5 also binds mature (spliced and aminoacylated) tRNAs and short non-coding dsRNAs in a Ran-GTP dependent mechanism , Wu, et al, 2015. Yeast strains lacking either Los1 or Msn5 alone revealed only moderate defects in the nuclear export of tRNAs, and the double knockout is still viable although it displays a strong synthetic defect in mature tRNA export (Takano, et al, 2005, Huang & Hopper, 2015, Wu, et al, 2015. This suggests that Los1 and Msn5 are not the only tRNA nuclear exporters.…”