Multiple Trans-Splicing Events Are Required to Produce a Mature Nadl Transcript in a Plant Mitochondrion

Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Multiple trans-splicing events are required to produce a mature nadl transcript in a plant mitochondrion Patricia L. Conklin, Robin K. Wilson, and Maureen R. Hanson Section of Genetics and Development, Cornell University, Ithaca, New York 14853 USA The mitochondrial gene encoding NADH dehydrogenase subunit 1 (had1) in Petunia hybrida is split into five exons, a, b, c, d, and e. With the use of a complete restriction map of the 443-kb Petunia mitochondrial genome, we have cloned these exons and mapped their location. Exon a is located 130 kb away from and in the opposite orientation from exons b and c. Exon d maps 95 kb away and in the opposite orientation from exons b and c. Exons d and e are separated by 190 kb. By performing the polymerase chain reaction on Petunia cDNAs, we have shown that transcripts from these five exons are joined via a series of cis- and trans-splicing events to create a mature had1 transcript. In addition, we have found 23 C -* U RNA edit sites in Petunia nadl. RNA editing changes 19 of the amino acids predicted by the genomic sequence. [Key Words: trans-splicing; nadl ; RNA editing; mitochondria; Petunia hybrida; introns] Received April 29, 1991; revised version accepted May 31, 1991. Several different RNA processing events can occur dur- NADH-dehydrogenase subunit I (had1) also contains in- ing the maturation of a protein-coding plant mitochon- trons. In contrast, nadl is encoded by a single open read- drial transcript. These events distinguish plant mito- ing frame (ORF) in the mammalian (Chomyn et al. 1985), chondria from their mammalian counterparts and in- Drosophila (Clary et al. 1984), and Chlamydomonas clude RNA editing, cis-splicing and, as we will describe (Boer and Gray 1988)mitochondrial genomes. In Neuro- here, trans-splicing. spora mitochondria, nadl is interrupted by a group I in- RNA editing has been shown to occur in many, if not tron (Burger and Werner 1985). Four exons that encode all, plant mitochondrial protein-coding genes; specific different regions of the NAD1 protein have been de- cytidines in the DNA sequence are altered in the RNA scribed in higher plant mitochondrial genomes. Two ex- and appear as thymidines in the cDNAs that are isolated ons that encode the central portion of NAD 1 were found from these genes (Covello and Gray 1989; Gualberto et in watermelon. These nadl exons are separated by a al. 1989; Hiesel et al. 1989). This is consistent with a group II intron of -1450 bp (Stern et al. 1986). The first process that alters cytidine into uridine residues, al- of these two exons was also described in tobacco and though uridine in place of cytidine in edited RNAs has maize (Bland et al. 1986). Two additional nadl exons, not been directly demonstrated. The extent of RNA ed- predicted to encode the carboxy-terminal portion of iting varies from gene to gene. Ten cytidines are edited in nadl, were recently reported in the broad bean mito- 75 codons of Petunia hybrida atp9 (Wintz and Hanson chondrial genome. Two group II intron-like domains 1991), whereas only 2 are edited within 99 codons of were found upstream of the first of these two exons. Oenothera rpsl4 (Schuster et al. 1990). RNA edits usu- Within the group II intron that separates these broad ally alter the protein sequence from that predicted from bean nadl exons, a maturase-like gene (mat-r) was the DNA sequence. found. The protein sequence predicted from this gene is Introns have been described in several plant mitochon- similiar to the maturases encoded within fungal mito- drial genes. A group II intron splits the coxlI gene in chondrial introns (Wahleithner et al. 1990). As yet, it is several, but not all, plant species that have been exam- not known whether the plant mat-r sequences actually ined (Fox and Leaver 1981; Hiesel and Brennicke 1983; specify a protein with maturase activity. Turano et al. 1987; Pruitt and Hanson 1989). The We commenced to clone the P. hybrida nadl exons Oenothera nad5-coding sequence is separated by two in- using probes from both watermelon and broad bean. In trons, one of which is clearly of the group II intron class addition to the exons described above, we cloned and (Wissinger et al. 1988). In maize (Hunt and Newton 1991) mapped an exon that encodes the amino-terminal por- and Petunia (C. Sutton et al., unpubl.), the proposed rps3 tion of NAD1 with the use of a probe from the equiva- gene also contains an intron. lent wheat exon (Chapdelaine and Bonen 1991). A com- The higher plant mitochondrial gene that encodes plete restriction map of the Petunia mitochondrial ge- GENES & DEVELOPMENT 5:1407-1415 9 1991by Cold Spring Harbor Laboratory Press ISSN 0890-9369/91 $3.00 1407 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Conklin et al. nome was constructed by Folkerts and Hanson (1989). terminal exon of Petunia to the nadl protein from hu- We were able to map the location and orientation of the man mitochondria (Anderson et al. 1981) made it evident nadl exons on the Petunia mitochondrial genome with that protein sequences in addition to those predicted by the use of this restriction mapping data. Our mapping the cloned Petunia exons were necessary to encode a data indicate that the five nadl exons are dispersed full-length NAD1 protein. A small region of protein se- around the 443-kb Petunia mitochondrial genome. With quence carboxy-terminal to exon c and the amino ter- the use of the polymerase chain reaction (PCR) on re- minus of NAD1 were not encoded by exons b and c and verse-transcribed mitochondrial RNA, we have shown the terminal exon (comparison not shown). that a series of trans- and cis-splicing events, in addition To obtain the Petunia nadl exon(s) that encode the to RNA editing, are necessary to create a mature nadl portion of the NAD1 protein between exon c and the transcript, nadl is the first example of a trans-spliced terminal exon, a PCR on reverse-transcribed transcripts mitochondrial gene. was undertaken. Two oligonucleotides were made as primers: The first was homologous to a sequence at the 5' end of exon b (primer b), and the second was homol- Results ogous to a sequence downstream of the terminal exon (primer e). The exact sequence and orientation of these Cloning the Petunia nadl exons primers is noted in Figure 1. A PCR amplification was Four of the five nadl exons encoded by the P. hybrida carried out on random hexamer-primed cDNAs from pu- line 3704 mitochondrial genome were found by virtue of rified suspension cell mitochondrial RNA. The amplifi- their similarity to nadl exons encoded by other higher cation product obtained using the exon b/e primer set plant mitochondrial genomes. Determination of the was -650 bp (data not shown). This PCR-amplified complete sequence of Petunia nadl was begun by clon- cDNA was subcloned and sequenced. A small exon (exon ing nadl exon c from a cosmid library of Petunia mito- d) was found spliced in-frame between exons c and the chondrial DNA with the use of a random hexamer-la- terminal exon (exon e). This small exon found in Petunia beled watermelon URF1 (exon c) probe, kindly provided is 98% identical at the DNA level to a small nadl exon by D. Stem. By restriction enzyme and sequence analy- found upstream of mat-r in broad bean (Wahleithner et sis, nadl exon b was found upstream of exon c, in an al. 1990). The sequence of Petunia nadl exon d is pre- arrangement very similiar to that of the analogous nadl sented in Figure 1. exons described in watermelon (Stern et al. 1986). The Between the Petunia nadl exon d and the mat-r se- sequence of Petunia nadl exons b and c, along with the quence, an unidentified ORF was found that is 96% iden- intron that separates them, is shown in Figure 1. The tical to the ORF85 found downstream of the equivalent DNA sequence of these Petunia nadl exons is 100% and exon in broad bean nadI (Wahleithner et al. 1990). The 99% identical to that of watermelon exons b and c pub- Petunia ORF (ORF85P) contains 92 codons in contrast lished previously (Stern et al. 1986). The sequence of the with the 85 codons in the broad bean ORF. 986-bp intron that separates exons b and c in Petunia is While the work in Petunia nadl was in progress, an quite similiar to the analogous intron in watermelon, exon (exon a), which encodes the amino terminus of except that a 459-bp insertion is present in the water- NAD1, was isolated in both the wheat (Chapdelaine and melon intron relative to Petunia. The position of this Bonen 1991) and Oenothera (Wissinger et al. 1991)mi- insertion is noted in Figure 1. tochondrial genomes. An oligonucleotide homologous to DNA probes from the broad bean terminal exon and the wheat exon a (sequence provided by L. Bonen)was the mat-r gene were then utilized to clone these coding used as a probe to clone Petunia nadl exon a from the regions from a Petunia mitochondrial cosmid library cosmid library of Petunia mitochondrial DNA. The se- with the help of D.R. Wolstenholme. The sequence of quence of the Petunia nadl exon a is shown in Figure 1. the Petunia nadl terminal exon (exon e) is shown in Restriction maps of the five Petunia nadl exons are Figure 1.

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