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

Hsiao-Yun Huang,Anita K Hopper

doi:10.1101/gad.258293.115

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.

Highlights

Eukaryotic tRNAs are transcribed in the nucleus but function in translation in the cytoplasm
Los1 interacts β-Importins in tRNA subcellular dynamics with both unspliced and spliced tRNAs, whereas we found no evidence for direct interaction of Mtr10 with tRNA
We developed in vivo β-importin complex co-IP assays to investigate in vivo interactions of β-importins with tRNAs

Summary

Introduction

Eukaryotic tRNAs are transcribed in the nucleus but function in translation in the cytoplasm. Cytoplasmic tRNAs are constitutively imported into the nucleus via retrograde tRNA nuclear import (Shaheen and Hopper 2005; Takano et al 2005; Zaitseva et al 2006; Shaheen et al 2007; Murthi et al 2010; Barhoom et al 2011; Miyagawa et al 2012). Because deletion of LOS1 results in the inhibition of primary tRNA nuclear export, end-processed intron-containing pre-tRNAs, which are unable to access the cytoplasmic splicing machinery, accumulate in los1Δ cells (Hopper et al 1980; Sarkar and Hopper 1998). Because tRNAs play essential roles in the cytoplasm, additional tRNA nuclear export pathways must exist, at least in yeast, Drosophila, and Arabidopsis

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Results

Conclusion