tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p͞ Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10͞Kap111 member of the importin-␤ family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.amino acid deprivation ͉ heterokaryon ͉ Mtr10 ͉ tRNA nuclear import ͉ yeast E ukaryotic precursor tRNAs (pretRNAs) contain 5Ј and 3Ј extra sequences and, for Ϸ25% of Saccharomyces cerevisiae (yeast) tRNA families, introns. To be fully functional, pretRNAs are subject to several maturation steps, including removal of the extra sequences, numerous nucleoside modifications, and addition of the 3Ј terminal CCA nucleotides (1). In vertebrates, nearly all tRNA posttranscriptional processing occurs in the nucleus before export of tRNAs to the cytoplasm (1-3). In contrast, several lines of evidence show that in yeast, pretRNA intron removal occurs in the cytoplasm, not the nucleoplasm. First, the subunits of the heterotetrameric tRNA-specific splicing endonuclease, Sen15, Sen2, Sen34, and Sen54, colocalize with mitochondria (4, 5). Tethering of a noncatalytic subunit, Sen54, to mitochondria does not inhibit pretRNA splicing, whereas mutations prohibiting its mitochondrial association cause accumulation of unspliced pretRNAs (4). Moreover, cells with conditional mutations in a gene encoding a catalytic subunit, Sen2, accumulate unspliced pretRNAs in the cytoplasm (4). A previous report employing immunofluorescence and immune electron microscopy concluded that a large fraction of tRNA ligase, catalyzing second step of pretRNA splicing, resides in the nucleus (6); however, more recent reports show that there is a functional pool of tRNA ligase activity in the cytoplasm (7) and that carboxyl GFP-tagged ligase is cytosolic (5). Finally, a carboxyl GFP-tagged version of the enzyme catalyzing the third step of pretRNA splicing, removal of the residual 2Ј phosphate (8), is distributed throughout the cytoplasm (5).Splicing of pretRNAs in the cytoplasm provides an explanation for the pretRNA splicing defects observed when there are mutations in the tRNA nuclear export machinery, including the tRNA exportin, Los1p (9-11), ...

No abstract

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support "feedback" of nucleus/cytosol exchange to the pre-tRNA splicing machinery.