Coordination of tRNA nuclear export with processing of tRNA

Abstract

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 59 and 39 ends, posttranscriptional addition of the 39 CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 59 and 39 ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t /tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 59 and 39 terminal ends, and the TCC loop are critical for exportin-t binding.

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…Studies of the binding capability of vertebrate exportin-t and crystallography studies of S. pombe Los1 in complex with tRNA and Ran-GTP showed that this exportin preferentially interacts with the appropriately structured tRNA backbone (the D and TcC loops of the L-shaped tertiary structure) as well as the mature tRNA 59 and 39 ends (Arts et al 1998b;Lipowsky et al 1999;Cook et al 2009;Lee et al 2011). Moreover, in vivo studies in S. cerevisiae demonstrated that tRNAs with altered sequence accumulate in the nucleus (Qiu et al 2000).…”

“…Although exportin-t/Los1 monitors the tRNA backbone and aminoacyl stem, it does not monitor the anticodon stem as vertebrate exportin-t binds intron-containing and spliced tRNAs with equal affinity (Arts et al 1998b;Lipowsky et al 1999). In yeast, Los1's in vivo interaction with introncontaining pre-tRNAs is evidenced by the fact that splicing in yeast occurs only after nuclear export on the outer surface of mitochondria and los1D cells accumulate intron-containing tRNA in the nucleus (Sarkar and Hopper 1998;Yoshihisa et al 2003Yoshihisa et al , 2007.…”

Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

“…In this case, newly synthesized aminoacyl-tRNA synthetases would enter the nucleus, either as free enzymes that assemble into a multienzyme complex or as a preformed complex. Synthetases would then associate with their cognate tRNAs, and multiple tRNAs and synthetases would be transported to the cytoplasm together, perhaps in cooperation with exportin-t (21,22) or EF1 (32). One advantage of such a model would be to maintain a cytoplasmic ratio of tRNA to cognate synthetase of 1:1 and would result in partial assembly of the cytoplasmic translation apparatus already in the nucleus.…”

“…Exclusion of defective tRNA from the cytoplasm was shown to be due to nuclear proofreading, and it was proposed that aminoacylation of tRNA serves as this proofreading step (18 -20). While there is still some discussion whether aminoacylation or binding to the nuclear export receptor, exportin-t, is actually responsible for proofreading, there is agreement that nuclear aminoacylation increases tRNA export efficiency (21,22).…”

P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics

“…The role of Los1 in tRNA export is conserved in many, but not all, organisms. Xpo-t, the Los1 vertebrate homolog, efficiently binds end-processed, intron-containing pre-tRNA and mature tRNA (Arts et al 1998b;Lipowsky et al 1999;Cook et al 2009). Orthologs of Los1 in plants (PAUSED) (Hunter et al 2003) and vertebrates (Xpo-t) (Arts et al 1998a;Kutay et al 1998) participate in tRNA nuclear export.…”