A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus

Abstract

We determined the complete mtDNA sequence of the centipede Lithobius forficatus and found that only one of the 22 inferred tRNA genes encodes a fully paired aminoacyl acceptor stem. The other 21 genes encode tRNAs with up to five mismatches in these stems, and some of these overlap extensively with the downstream genes. Because a well-paired acceptor stem is required for proper tRNA functioning, RNA editing in the products of these genes was suspected. We investigated this hypothesis by studying cDNA sequences from eight tRNAs and found the editing of up to 5 nt at their 3' ends. This editing appears to occur by a novel mechanism with the 5' end of the acceptor stem being used as a template for the de novo synthesis of the 3' end, presumably by an RNA-dependent RNA polymerase. In addition, unusual secondary structures for several tRNAs were found, including those lacking a TPsiC (T) or a dihydrouridine (D) arm, and having an unusual number of base pairs in the acceptor or anticodon stems.

Figure 1

Gene map of L. forficatus mtDNA. Protein and rRNA genes are abbreviated as follows: atp6 and atp8 (genes for subunits 6 and 8 of the F₀ ATPase), cox1–cox3 (genes for cytochrome c oxidase subunits 1–3), cob (gene for apocytochrome b), nad1–nad6 and nad4L (genes for NADH dehydrogenase subunits 1–6 and 4L), and rrnS and rrnL (genes for small and large subunit rRNAs). The 22 tRNA genes are identified by the one-letter code for the corresponding amino acid. Two leucine and two serine tRNA genes are differentiated by their anticodon sequence with trnL(uag) marked as L1, trnL(uaa) as L2, trnS(ucu) as S1, and trnS(uga) as S2. The direction of transcription for each gene is shown by an arrow. trnI (shaded area) is the only gene translocated relative to the arthropod primitive gene arrangement exemplified by the mtDNA of the horseshoe crab Limulus polyphemus (10). The numerals near each tRNA gene indicate the number of mismatches in the acceptor stems inferred from mtDNA sequence.

Figure 2

Primary and secondary structures of L. forficatus mitochondrial tRNAs. (A) Inferred from mtDNA sequence. (B) Inferred from cDNA sequences. Edited nucleotides are in boldface type and base pairs that are not present in all clones are in parentheses. Lines connecting tRNAs for R, N, S1, and E in A indicate that this is contiguous genomic sequence. Lines connecting the 5′ and 3′ ends of the tRNAs in B indicate bonds made by the RNA ligase during cDNA preparation.

Figure 3

Alternative foldings for the unedited L. forficatus tRNAs with unusual secondary structures. Arrows indicate the deduced processing at the boundaries of these structures as observed in cDNA.