The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

Abstract

The import of nuclear-encoded RNAs into mitochondria is required for proper mitochondrial function in most organisms. However, the mechanisms used to achieve RNA import are largely unknown. In particular, the RNA elements that direct import have not been identified in any organism. In Tetrahymena, only one of three nuclear-encoded glutamine accepting tRNAs is imported into mitochondria. We transform Tetrahymena with marked glutamine tRNAs and quantitate their level of accumulation in mitochondria. Of several isostructural nucleotide substitutions tested, alteration of the anticodon sequence uniquely abolishes import. Furthermore, substitution of a single anticodon nucleotide (UUA-,UUG) confers import on a normally nonimported glutamine tRNA. Thus, the anticodon functions as a mitochondrial localization signal and is both necessary and sufficient for tRNA import. Given the prior evidence that neither the cytoplasmic nor the mitochondrial glutaminyl-tRNA synthetase distinguishes between the imported and nonimported glutamine tRNAs with respect to aminoacylation, we propose that some mitochondrial import factor distinct from a synthetase recognizes the anticodon of the imported glutamine tRNA.

The RNase P Associated with HeLa Cell Mitochondria Contains an Essential RNA Component Identical in Sequence to That of the Nuclear RNase P

“…The occurrence in mitochondria of nucleus-encoded RNA species has been previously reported, for tRNAs, in plants (59), Tetrahymena thermophila (53,54), Leishmania (1), Trypanosoma brucei (24), and S. cerevisiae (38), for MRP RNA, in mouse L cells (11) and mouse cardiomyocytes and myogenic cells (34), and, more recently, for 5S rRNA, in mammalian cells (36). In addition human immunodeficiency virus RNA has been detected in mitochondria of infected cells (60).…”

“…Thus, it has been shown that in S. cerevisiae, the nucleus-encoded tRNA Lys(CUU) is unequally distributed between the cytosol (95%) and mitochondria (5%) (18). Furthermore, in T. thermophila, ϳ10% of the nucleus-encoded tRNA Gln(UUG) is imported into mitochondria while the rest functions in the cytosol (53,54).…”

Mutations in a tRNA Import Signal Define Distinct Receptors at the Two Membranes of Leishmania Mitochondria

“…There is also evidence that other domains of the tRNA molecule besides the D arm contain import signals. In tetrahymena, the anticodon of tRNA Gln (UUG) functions as an import signal (20). Moreover, a tRNA Ile derivative containing a nonfunctional D arm sequence from tRNA Gln is nonetheless imported into leishmania mitochondria in vivo (10), indicating the presence of a signal elsewhere in the molecule, possibly in the anticodon arm, in addition to the one in the D arm.…”

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

“…Mitochondrial import of cytoplasmic tRNAs has been demonstrated in plants, fungi, protozoa, and mammals (Schneider et al+, 1994;Hauser & Schneider, 1995;Dietrich et al+, 1996;Entelis et al+, 1996;MarechalDrouard et al+, 1996;Tarassov & Martin, 1996)+ The number of imported tRNAs varies from a complete set necessary for translation in kinetoplastid protozoa (Simpson et al+, 1989;Hancock & Hajduk, 1990;Lye et al+, 1993;Chen et al+, 1994;Shi et al+, 1994) to 19-26 tRNAs in the ciliate protozoa (Suyama, 1986;Pritchard et al+, 1990), to one of three lysine isoacceptors in Saccharomyces cerevisiae (Entelis et al+, 1996)+ Importation of tRNA in yeast and Tetrahymena (Rusconi & Cech, 1996a, 1996b) is a very specific process capable of discriminating one particular tRNA out of a set of isoacceptor tRNAs+ In the case of the trypanosomatids, the importation system may have a broader specificity due to the fact that all mitochondrial tRNAs are imported+…”

“…The mechanism by which negatively charged RNAs cross the hydrophobic environment of the organelle's membranes remains a complete mystery, with apparent differences between species+ Importation of tRNA Lys (CUU) into the yeast mitochondrion is energy dependent and requires the preprotein import machinery as well as the presence of the precursor form of the mitochondrial lysyl-tRNA synthetase and also requires the tRNA to be aminoacylated (Tarassov et al+, 1995a;Entelis et al+, 1996Entelis et al+, , 1998)+ In trypanosomatids, mutated tRNAs that cannot be aminoacylated can still be imported in vivo (Schneider et al+, 1994) + Mahapatra et al+ (1998) have shown that no cytosolic factors are required for importation of synthetic tRNA into isolated Leishmania tropica mitochondria+ Two Leishmania mitochondrial surface proteins have been reported to bind RNA in the absence of ATP, but their precise role and identity remain to be determined (Adhya et al+, 1997) + Mahapatra et al+ (1998) have also proposed that the sequence motif AUGGCAGAG in the D-loop region of Leishmania tRNAs serves as the signal for import+ However, a recent analysis (Suyama et al+, 1998) of all available trypanosomatid tRNA sequences found no apparent consensus sequences within the D-loop that could account for regulation of tRNA importation+ It has become apparent that the tRNA molecule itself bears structural features required and sufficient for mitochondrial localization+ However, the precise features required appear to vary among different organisms: the anticodon in Tetrahymena (Rusconi & Cech, 1996a, 1996b, the D-arm in Leishmania (Lima & Simpson, 1996;Mahapatra et al+, 1998), and the anticodon arm and acceptor stem in yeast (Entelis et al+, 1998;Kazakova et al+, 1999)+ In this article, we demonstrate a selective ATPdependent importation of in vitro-transcribed tRNAs and several small synthetic RNAs into isolated mitochondria from L. tarentolae+…”