FIG. 4.
Mutation of the YGG(C/A)RRC motif in the 5′ flanking sequence of tRNALeu* influences localization of tRNALeu* and other mitochondrially localized tRNAs. (A) Black boxes in pSer-Leu* represent the sequence element GGCGG, which is 4 nucleotides upstream of tRNALeu* and directly abuts tRNASer. Open boxes in pSer-Leu* 1 and pSer-Leu* 2 denote mutations of the GGCGG sequence element to UCGCU. (B) Primer extension analysis of pSer-Leu*, pSer-Leu* 1, and pSer-Leu* 2 was performed with oligonucleotide LE4 on 1 μg of mitochondrial (M) or cytosolic (C) RNA. The endogenous tRNALeu is detected by a 25-nucleotide extension product, and the tagged tRNALeu* is detected by a 28-nucleotide product. (C) Localization of endogenous tRNAMet-e was determined by primer extension analysis with oligonucleotide Met e in pSer-Leu*, pSer-Leu* 1, and pSer-Leu* 2. (D) Primer extension analysis with oligonucleotide Met i shows the localization of tRNAMet-i in pSer-Leu*, pSer-Leu* 1, and pSer-Leu* 2. (E) The ratio of mitochondrial/cytosolic RNA for tRNALeu*, endogenous tRNALeu, and tRNAMet-e was analyzed for each transfectant. This ratio was set to an import level of 1 for tRNAs from pSer-Leu*, and the ratios for tRNAs from pSer-Leu* 1 and pSer-Leu* 2 were compared to that from pSer-Leu* in graphical form (n = 6 for tRNALeu* and tRNALeu; n = 4 for tRNAMet-e). (F) A poison primer extension assay was performed on 1 μg of a mitochondrial (M) or cytosolic (C) RNA fraction from the transfectant lines pSer-Leu*, pSer-Leu* 1, and pSer-Leu* 2. Oligonucleotide 9Sb produces a 22-nucleotide extension product, predominantly in the mitochondrial fraction. Oligonucleotide SL2 also produces a 26-nucleotide extension product, but it is primarily in the cytosol. Mitochondrial or cytosolic fractions with >3% cross-contamination were not used for subsequent experiments. SL RNA, spliced leader RNA.