Proc Natl Acad Sci U S A 97(25): 13738-13742

A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede <em>Lithobius forficatus</em>

Department of Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109-1048

^{To whom reprint requests should be addressed. E-mail:}ude.hcimu@vorvald.

^{Present address: Department of Energy's Joint Genome Institute, 2800 Mitchell Drive, Building 100, Walnut Creek, CA 94598.}

Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved October 11, 2000

Received 2000 Aug 21

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 TΨC (T) or a dihydrouridine (D) arm, and having an unusual number of base pairs in the acceptor or anticodon stems.

Abstract

RNA editing has been defined as “any programmed alteration of RNA primary structure to generate a sequence that could have been directly encoded at the DNA (gene) level” (1). First discovered less than 15 years ago, it is now regarded as a widespread phenomenon occurring in all types of eukaryotic RNA, mainly in mitochondria, less frequently in chloroplasts, and, in a few cases, in the nucleus (2). Four different types of tRNA editing have been found to date (all of them in mitochondria): cytidine to uridine conversion, cytidine or uridine insertion, template-dependent editing of the first three nucleotides at the 5′ ends of tRNAs, and template-independent editing at the 3′ ends of tRNAs (for review, see ref. 3). With a single exception (4), only the last type has been reported so far in animal tRNAs. None of the previously described types of tRNA editing, however, could serve as a potential editing mechanism for the aberrant tRNA structures encoded by the mtDNA of the centipede Lithobius forficatus.

We have sequenced the complete mtDNA of L. forficatus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF309492","term_id":"11692757"}}AF309492) and found that all but one of its 22 tRNA genes code for tRNAs with at least one and as many as five mismatches in their aminoacyl acceptor stems (henceforth termed acceptor stems). Some, but not all, of these tRNA genes, if they encode full-length acceptor stems, would also overlap with their downstream genes. Because a well-matched acceptor stem is important for defining tRNA structure (5), directing tRNA processing (6), and assisting tRNA recognition by aminoacyl-tRNA synthetase (7), we suspected that the poorly matched acceptor stems of L. forficatus tRNAs are posttranscriptionally edited. We studied this hypothesis by analyzing cDNA sequences from eight tRNAs and found that a previously unknown type of tRNA editing is present in the mitochondria of L. forficatus.

Underlined nucleotides are part of the primer sequence. Nucleotides in boldface are those inferred to be edited at the 3′end of the acceptor stem. Nucleotides in italics are inferred to be added by some alternative (nontemplated) editing process(es). Nucleotides in lowercase are inferred to be artifacts of the experimental procedure. D indicates nucleotide variation (A, T, or G) among the four edited cDNA clones for tRNA (R), possibly caused by posttranscriptional modification at this tRNA position (26).

Acknowledgments

We thank K. G. Helfenbein and J. V. Moran for comments on the manuscript. This work was supported by National Science Foundation Dissertation Improvement Grant DEB 9972712 to W.M.B. and D.V.L., National Science Foundation Grant DEB 9807100 to W.M.B. and J.L.B., and a University of Michigan predoctoral fellowship to D.V.L.

Acknowledgments

Abbreviation

RdRp	RNA-dependent RNA polymerase

Abbreviation

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF309492","term_id":"11692757"}}AF309492).

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.250402997.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.250402997

Footnotes

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