Characterization and localization of mitochondrial DNA-encoded tRNAs and nuclear DNA-encoded tRNAs in the sea anemone Metridium senile

• Published in: Current genetics Vol. 59; no. 3; pp. 139 - 152
• Main Authors: Beagley, C. Timothy, Wolstenholme, David R
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.08.2013; Springer Berlin Heidelberg; Springer Nature B.V
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biochemistry; Biomedical and Life Sciences; Cell Biology; Cell Nucleus - genetics; Cytoplasm - genetics; Cytoplasm - metabolism; DNA - genetics; DNA primers; DNA, Mitochondrial - genetics; genes; Life Sciences; Metridium senile; Microbial Genetics and Genomics; Microbiology; Mitochondria - genetics; Mitochondria - metabolism; Mitochondrial DNA; Northern blotting; Nucleic Acid Conformation; nucleotide sequences; Plant Sciences; Protein synthesis; Proteomics; Research Article; RNA, Transfer - biosynthesis; RNA, Transfer - chemistry; RNA, Transfer - genetics; Sea Anemones - genetics; transfer RNA; CCA addition; Importation; Mitochondrial tRNAs; Nuclear tRNAs; tRNA genes
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/s00294-013-0395-9

The mitochondrial (mt) genome of the sea anemone Metridium senile contains genes for only two transfer RNAs (tRNAs), tRNAf-Met and tRNATrp. Experiments were conducted to seek evidence for the occurrence of functional tRNAs corresponding to these genes and for the participation of nuclear DNA-encoded tRNAs in mt-protein synthesis. RNA sequences corresponding to the two mt-tRNA genes were located in mitochondria and it was shown that 3′-CC (and possibly A, but no other nucleotide) is added post-transcriptionally to the 3′ end of at least 50 % of mt-tRNAf-Met molecules and to a small fraction of the mt-tRNATrp molecules. Using specific oligonucleotide primers based on expected nuclear DNA-encoded tRNAs in a series of RACE experiments, we located the nuclear genes for tRNAGln, tRNAIle, tRNAi-Met, tRNAVal and tRNAThr. Data from Northern blot analyses indicated that mtDNA-encoded tRNAf-Met is limited to mitochondria but that nuclear DNA-encoded tRNAVal and tRNAi-Met are present in the cytoplasm and in mitochondria. These data provide direct evidence that in M. senile, mature, functional tRNAs are transcribed from the mtDNA-encoded tRNAf-Met and tRNATrp genes, and are consistent with the interpretation that both nuclear DNA-encoded tRNAVal and tRNAi-Met are utilized in mitochondrial and cytosolic protein synthesis.