Abstract
A yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay based on amber suppression was used to identify proteins that participate in the nuclear tRNA export process in Saccharomyces cerevisiae. One of the proteins identified by this strategy is Utp8p, an essential 80-kDa nucleolar protein that has been implicated in 18 S ribosomal RNA biogenesis. Our characterization indicated that the major function of Utp8p is in nuclear tRNA export. Like the S. cerevisiae Los1p and the mammalian exportin-t, which are proteins known to facilitate nuclear tRNA export, overexpression of Utp8p restored export of tRNAamTyr mutants defective in nuclear export. Furthermore, depletion of Utp8p blocked nuclear export of mature tRNAs derived from both intronless and intron-containing pre-tRNAs but did not affect tRNA and rRNA maturation, nuclear export of mRNA and ribosomes, or nuclear tRNA aminoacylation. Overexpression of Utp8p also alleviated nuclear retention of non-aminoacylated tRNATyr in a tyrosyl-tRNA synthetase mutant strain. Utp8p binds tRNA directly and saturably, indicating that it has a tRNA-binding site. Utp8p does not appear to function as a tRNA export receptor, because it does not shuttle between the nucleus and the cytoplasm. Taken together, the results suggest that Utp8p is an essential intranuclear component of the nuclear tRNA export machinery, which may channel tRNA to the various tRNA export pathways operating in S. cerevisiae.
Highlights
Translocation of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)1 located in the nuclear envelope
The ATP (CTP):nucleotidyltransferase (Cca1p) is an essential enzyme that prepares tRNAs for aminoacylation in the nucleus, cytoplasm, and mitochondrion by adding the nucleotides C, C, and A to the 3Ј ends of tRNAs. This maturation step, but not aminoacylation itself, appears to be absolutely required for nuclear export of tRNAs in both mammalian cells and S. cerevisiae, and because it has been shown that exportin-t preferentially binds tRNAs with the 3Ј CCA ends, nuclear tRNA export is blocked in a cca1 S. cerevisiae mutant strain, and tRNAs lacking CCA were not exported to the cytoplasm in X. laevis [12, 14, 16, 17, 20]
Identification of Utp8p Using a Yeast tRNA Three-hybrid Selection System—To identify proteins that participate in the nuclear tRNA export process in S. cerevisiae, a yeast tRNA three-hybrid selection approach was used to screen a cDNA library for genes encoding proteins that interact with tRNA (Fig. 1)
Summary
Like methionyl-tRNA synthetase, overexpression of Cca1p restored nuclear export of tRNAMet, a tRNA made from intronless pre-tRNA, in the los mutant strain, and the protein was shown to shuttle between the nucleus and cytoplasm [19] These results led to the suggestion that Cca1p may function as a tRNA export receptor or an adaptor in a Los1p- and nuclear aminoacylation-independent pathway that is required for export of tRNAs obtained from intronless pre-tRNAs [19]. This is consistent with the finding that loss of Los1p function and nuclear tRNA aminoacylation did not affect the viability of the cells [18] It is not known whether Cca1p or another unidentified Los1p- and aminoacylation-independent pathway facilitates nuclear export of tRNAs derived from intron-containing pre-tRNAs. The genetic and biochemical studies reported suggest that nuclear tRNA export in S. cerevisiae involves multiple redundant pathways. Utp8p may serve as an intranuclear factor that delivers aminoacylated and non-aminoacylated tRNAs to the appropriate tRNA export pathway
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