Edited transcripts compete with unedited mRNAs for trans-acting editing factors in higher plant chloroplasts Martha L. Reed, Sangbom M. Lyi, Maureen R. Hanson * Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA Received 12 February 2001; received in revised form 14 May 2001; accepted 29 May 2001 Received by D. Baulcombe Abstract Chloroplast RNA transcripts of vascular plants undergo C to U editing at approximately 30 sites, but there is no consensus sequence that identi®es a C to be edited. Both sequences closely surrounding an edited C and unidenti®ed site-speci®c trans-acting factors have been shown to be important for editing. The ability of an already edited transgenic sequence to bind and thus titrate a trans-acting editing factor was evaluated for two editing sites, ndhF and rpoB site 2. The U-containing rpoB transcripts did not affect editing of the endogenous rpoB transcripts, likely because the comparable C-containing transcripts containing 27 nucleotides surrounding the edited C were only 20% edited, indicating a low af®nity of a trans-factor for this length of edited sequence. Surprisingly, U-containing ndhF transgene transcripts reduced endogenous ndhF transcript editing to the same degree as a C-containing transgene transcript. This indicates that the C target of editing is not a critical recognition feature for the site-speci®c trans-acting factor. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Chloroplast; Transgenic plant; Gene expression; ndhF; rpoB 1. Introduction Chloroplast mRNAs of vascular plants undergo modi®ca- tion by RNA editing (reviewed in Gray and Covello, 1993; Hanson et al., 1996; Maier et al., 1996; Smith et al., 1997; Bock, 2000). In those dicots and monocots that have been studied, 20±30 different Cs are altered to Us (Maier et al., 1995; Hirose et al., 1999; Corneille et al., 2000). A typical edited mRNA carries only a few C targets of editing, usually one to six. Most chloroplast RNA editing occurs in the second codon position, altering the predicted amino acid to one more conserved relative to those encoded by homo- logous genes in other organisms. The mechanism by which the editing apparatus selects C targets of editing is unknown. There are no consensus sequences or obvious secondary structures speci®c to the sequences surrounding edited Cs that can predict the loca- tion of the editing target. Nevertheless, sequences immedi- ately surrounding the edited C have clearly been demonstrated to be important for editing. Small chimeric chloroplast RNAs containing short segments of sequences surrounding an edited C have been found to serve as editing substrates in transgenic plants in vivo (Chaudhuri and Maliga, 1996; Bock et al., 1996; Reed and Hanson, 1997). All such experiments reported to date have been carried out in tobacco, whose chloroplasts are the most readily trans- formed by current methods. Such experiments have estab- lished that the requirements for length of surrounding sequence for in vivo editing varies somewhat depending on the editing site being tested, but that for some sites such as psbL, less than 30 nt of surrounding sequences is adequate to specify editing of the transgene transcripts (Chaudhuri et al., 1995). Moreover, experiments have demonstrated that single nucleotide alterations in sequences surrounding editing sites expressed from transgenes can greatly reduce the extent of editing of the transgene tran- scripts (Chaudhuri and Maliga, 1996; Bock et al., 1997; Reed et al., 2001). None of the components of the editing apparatus have been identi®ed at the molecular level. However, indirect evidence of site-speci®c trans-acting factors has emerged from experiments designed to study cis-acting elements in transgenic chloroplasts. When transgenes carrying either psbL, ndhD, or rpoB sequences were introduced into chlor- Gene 272 (2001) 165±171 0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S0378-1119(01)00545-5 www.elsevier.com/locate/gene Abbreviations: aadA, aminoglycoside adenyl transferase; CTAB, hexa- decyltrimethylammonium bromide; GFP, green ¯uorescent protein; MOPS, morpholinepropanesulfonic; ndhF, NADH dehydrogenase subunit F; nt, nucleotide; PCR, polymerase chain reaction; rpoB, RNA polymerase subu- nit B; RT-PCR, reverse transcription PCR * Corresponding author. Tel.: 11-607-254-4833/4832; fax: 11-607-255- 6249. E-mail address: mrh5@cornell.edu (M.R. Hanson).