Evidence for import of a lysyl-tRNA into marsupial mitochondria - PubMed (original) (raw)

Evidence for import of a lysyl-tRNA into marsupial mitochondria

M Dörner et al. Mol Biol Cell. 2001 Sep.

Free PMC article

Abstract

The mitochondrial tRNA gene for lysine was analyzed in 11 different marsupial mammals. Whereas its location is conserved when compared with other vertebrate mitochondrial genomes, its primary sequence and inferred secondary structure are highly unusual and variable. For example, eight species lack the expected anticodon. Because the corresponding transcripts are not altered by any RNA-editing mechanism, the lysyl-tRNA gene seems to represent a mitochondrial pseudogene. Purification of marsupial mitochondria and in vitro aminoacylation of isolated tRNAs with lysine, followed by analysis of aminoacylated tRNAs, show that a nuclear-encoded tRNA(Lys) is associated with marsupial mitochondria. We conclude that a functional tRNA(Lys) encoded in the nuclear genome is imported into mitochondria in marsupials. Thus, tRNA import is not restricted to plant, yeast, and protozoan mitochondria but also occurs also in mammals.

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Figures

Figure 1

Figure 1

(A) Alignment of mitochondrial lysyl-tRNA genes from 11 marsupials. The D. virginiana sequence served as a reference. Dots indicate identity to the D. virginiana sequence, dashes indicate deletions, and substitutions are given by the individual nucleotide. The inferred anticodon is printed in red. Secondary tRNA structure elements are indicated above the alignment. (B) Inferred secondary structures of mitochondrial lysyl-tRNAs from_M. domestica_ (anticodon UUU), S. crassicaudata (UCU), M. robustus (ACA),M. rufus (AUA), and human (UUU). Indicated positions 9, 33, 37, and 73 are described in the text. Numbering of the positions is according to Sprinzl et al. (1998) and does not correspond to the actual position in the tRNA. Note that the anticodon-loop of lysyl-tRNA from S. crassicaudata consists of eight instead of seven nucleotides.

Figure 2

Figure 2

Southern blot hybridization of_Bgl_II-, Eco_RI-, and_Pvu_II-digested total cellular DNA from D. virginiana and mouse. (A) Hybridization with radiolabeled oligonucleotide mt. K-cons. for the placental type of mitochondrial lysyl-tRNA. (B) Hybridization with oligonucleotide mt. K-op for_D. virginiana mitochondrial lysyl-tRNA. Signals in A and B correspond to restriction fragments containing the individual mitochondrial gene.

Figure 3

Figure 3

Northern blot hybridization of total cellular RNA from D. virginiana and mouse. E. coli tRNAs are included to serve as hybridization background controls. (A) Hybridization with oligonucleotide mt. K-op for D. virginiana mitochondrial lysyl-tRNA. (B) Hybridization with radiolabeled oligonucleotide mt. K-cons. for the placental type of mitochondrial lysyl-tRNA.

Figure 4

Figure 4

(A) Secondary structure drawing of mitochondrial tRNALys from M. domestica within the precursor transcript. Scissors indicate 5′- and 3′-processing cleavage positions as determined from cDNA clone analysis after circularization or tagging of the tRNA. (B) Posttranscriptional addition of the CCA terminus and further 3′-terminal extensions (shown in red). Numbers indicate the frequency at which individual clones were observed.

Figure 5

Figure 5

Strategy to identify the nature of the mitochondrial lysyl-tRNA. tRNAs were isolated from highly purified mitoplasts from M. domestica and deacylated (green dots indicate lysine; red dots denote any amino acid except lysine). tRNAs were charged with lysine (green dot) on incubation with a mitochondrial S100 extract. The 3′-OH groups of nonaminoacylated tRNAs were blocked by oxidation with the use of NaIO4 (red X). Subsequently, the tRNA protected by lysine was deacylated, leading to a free 3′-OH group that can be selectively labeled by pCp ligation.

Figure 6

Figure 6

(A) In vitro aminoacylation of deacylated mitochondrial tRNA fractions from M. domestica with lysine, followed by periodate oxidation of nonprotected molecules. Lane nox designates mitochondrial tRNAs that were labeled without prior aminoacylation or oxidation. Lane ox indicates that mitochondrial tRNAs were oxidized quantitatively with a 1000-fold molar excess of NaIO4 so that no molecules could be labeled at the 3′-end. Mitochondrial tRNA fraction of lane K was aminoacylated in vitro with lysine before oxidation. Aminoacylated, oxidation-resistant tRNAs were deacylated and 3′-end labeled. The band indicated by the asterisk was isolated for sequence analysis (two reactions) and identified as a nuclear type of tRNALys(TTT). (B) Alignment of the human nuclear tRNALys and the obtained sequence of the marsupial tRNALys associated with mitochondria. The alignment was done according to Corpet (1988). Human cyt, human cytosolic tRNALys; Monodelphis mt, mitochondria-associated tRNALys in Monodelphis; Y = C or U; W = A or U.

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