Evolutionary and functional aspects of C-to-U editing at position 28 of tRNACys(GCA) in plant mitochondria

In plant mitochondria, editing of messenger RNA by C-to-U conversions is essential for correct gene expression as it usually improves the protein-sequence conservation between different species or sometimes affects the reading frames (for a review, see Maier et al+, 1996)+ Editing sites have been identified in mitochondrial (mt) RNA of all major groups of land plants, including Bryophytes, Pteridophytes, Prespermaphytes, and Spermaphytes (Hiesel et al+, 1994a,b;Malek et al+, 1996)+ Editing mainly affects messenger RNA, but editing sites have also been identified in three transfer RNAs+ In dicot mitochondria a C-to-U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of tRNAPhe(GAA) (Marechal-Drouard et al+, 1993; Binder et al+, 1994)+ In the gymnosperm Larix leptoeuropaea, three C-to-U conversions restore a U:A base pair in the acceptor stem, D stem, and anticodon stem of tRNAHis(GUG), respectively (Marechal-Drouard et al+, 1996b)+ The third example described is the Oenothera berteriana mt tRNACys(GCA), where a C28:U42 mismatch is converted into a U28:U42 noncanonical base pair (Binder et al+, 1994)+ In the case of both tRNAPhe and tRNAHis, editing of precursors is a prerequisite for 59 and 39 processing to generate a mature tRNA (Marchfelder et al+, 1996; Marechal-Drouard et al+, 1996a, 1996b; Kunzmann et al+, 1998)+ The role of editing in the case of tRNACys has not been studied so far, although it has been shown that it occurs at the precursor level (Binder et al+, 1994)+ In this letter, we report an evolutionary and functional study of mt tRNACys(GCA) editing in plant mitochondria+ The cloverleaf structure of the mt tRNACys(GCA) deduced from the sequence of the single Solanum tuberosum mt trnC gene (EMBL Accession Number X93575) is identical to its counterpart in O. berteriana and reveals a weak anticodon stem with a U27:G43 noncanonical interaction, and a C28:U42 mismatch+ By analyzing RT-PCR amplified cDNAs of S. tuberosum mt tRNACys precursors (362 nt in length), we found that 7 out of 11 independent clones contained a T at position 28+ The ratio of edited versus nonedited mature tRNACys was determined by RT-mini-sequencing+ When total S. tuberosum mt tRNAs were used as template, only dATP was incorporated, demonstrating that the mature tRNACys is fully edited in vivo (Fig+ 1B)+ From an evolutionary point of view, the comparison of the S. tuberosum mt trnC gene with its counterpart in Marchantia polymorpha shows in particular two differences in the anticodon stem (Fig+ 1A)+ In M. polymorpha, an A residue at position 43 allows a T27:A43 base pairing, and a T residue is present at position 28+ Considering that this sequence is more closely related to the ancestral sequence, we postulated that the C-to-U editing site found in dicot mitochondria restores this ancestral sequence+ To confirm this hypothesis, we first tried to determine when, during the evolution of land plants, the mt trnC gene acquired a C at position 28 and when the C28-to-U28 editing event occurred+ To do so, the internal sequence of trnC (from position 25 to 52) was PCR-amplified, cloned, and sequenced in several species that belong to different groups of land plants+ A single difference could be observed in this region between the different plants tested: a T residue was present at position 28 of mt trnC in the Pteridophyte Pteris nephrolepis (Filicales order) and in the Reprint requests to: Laurence Marechal-Drouard, Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique, Universite Louis Pasteur, 12 rue du General Zimmer, F-67084 Strasbourg Cedex, France; e-mail: laurence+drouard@ibmpulp+u-strasbg+fr 2Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801, USA 3Present address: Department of Molecular Biophysics and Biochemistry, Yale University, School of Medicine, New Haven, Connecticut 06520-8024, USA RNA (2000), 6:470–474+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+