Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5175-5179, July 1986 Genetics Autonomously replicating RNA in mitochondria of maize plants with S-type cytoplasm (cytoplasmnic male sterility/genetic RNA/RNA plasmids/double-stranded RNA/in organello RNA synthesis) PATRICK M. FINNEGAN AND GREGORY G. BROWN Centre for Plant Molecular Biology, Department of Biology, McGill University, Montreal, PQ, Canada H3A iB1 Communicated by Hewson Swift, March 17, 1986 ABSTRACT Mitochondria isolated from maize plants with in organello RNA synthesis system to search for differences S-type male-sterile cytoplasms are capable of synthesizing four in mitochondrial gene expression among the various maize species of RNA at concentrations of actinomycin D that cytoplasmic types. We have made the surprising discovery eliminate all DNA-directed RNA synthesis. No RNA synthesis that several R$As unique to the S cytoplasm are synthesized occurs under the same conditions with mitochondria from in a DNA-independent manner. Our observations indicate plants possessing normal (N) cytoplasm or with other that these RNAs represent an RNA-based genetic system subcellular fractions from plants with S cytoplasm. The that is endogenous to the mitochondnron. No other such actinomycin D-resistant RNA synthesis occurs within the system has been found in an organelle. Because these RNAs mitochondria since the labeling ofthese species is unaffected by replicate independently of cellular chromosomes, we have inclusion of RNase in the incubation medium and since they termed them RNA plasmids. become completely sensitive to RNase upon lysis of the mitochondria with low concentrations of Triton'X-100. Two of the actinomycin D-resistant products are double stranded. MATERIALS AND METHODS These are 2850 and 900 base pairs in length, whereas the remaining two are 2150 and 850 bases. The synthesis ofall four Materials. Inbred WF9 seeds were from Marc Albertsen RNAs occurs in at least five different accessions ofS cytoplasm, (Pioneer Hi-Bred International, Johnston, IA); inbred B73 suggesting it is a general feature of S qitochondria. The seeds were from Mike Brayton Seeds (Ames, IA); Vg double-stranded RNAs show homology to single-stranded S cytoplasm seed (open-pollinated) was from C. S. Levings III mitochondrial RNA but not to N niutochondrial RNA. Our (North Carolina State University, Raleigh, NC). Miracloth is observations indicate that the replication of these RNAs occurs a product of Calbiochem. [a-32P]UTP (410 or 3000 Ci/mmol, independently of mtDNA and that they thus represent a novel 1 Ci = 37 GBq) was obtained from Amersham. Percoll was type of inheritable element in organelles, an RNA plasmid. from Pharmacia, CF-li cellulose was from Whatman, and GeneScreen transfer membrane is a product of New England Many viruses store genetic information as RNA, but such Nuclear. genetic RNA is rarely encountered in uninfected cells. In the Mitochondrial Isolation and Cell Fractionation. Mitochon- only well-documented examples in which genetic RNA is dria were prepared from 5- or 6-day-old etiolated maize found as a normal cellular constituent, it is associated with shoots and roots essentially as described (10). Extramito- virus-like capsid structures in the cytosol, as in the cases of chondrial RNA was removed by treating the mitochondria, the killer plasmid ofyeast (1), the virus-like particles ofother suspended in homogenization buffer, with 10 pug of RNase A fungi (2), and the cryptic viruses ofplants (3). In each case the per g of starting material for 60 min. The mitochondria were capsid-associated RNA is usually double stranded, and collected from the reaction mixture by centrifugation through replicative and messenger RNA synthesis takes place using a cushion of 2 vol of 0.6 M sucrose/10 mM N-[tris(hydroxy- an RNA template. methyl)methyl]aminoethanesulfonic acid (Tes)/20 mM The mitochondrial genomes of higher plants possess a EDTA (sucrose buffer) at 10,000 x g for 20 min before further number of features that are not found in nuclear, viral, or purification by centrifugation through 20-60% sucrose gra- other organelle genomes. They exist as a set of large dients (11). The purified mitochondria were slowly diluted recombining circular molecules in which some circles are over 15 min with sucrose buffer before collection by centrif- ugation at 12,000 x g for 10 min. The pellet fraction of the more abundant than others, resulting in unequal representa- sucrose-gradient centrifugation consisted mainly of nuclear tion of the various sequences that they comprise (4, 5). contamination and is referred to as the nuclear pellet. The Minicircular plasmid DNAs are also frequently present in pellet resulting from the first centrifugation of the tissue plant mitochondria (6) and linear DNA episomes that recom- homogenate is referred to as the 1000 x g pellet. Microsome bine with specific sequences on the large circular molecules and cytosol fractions are the pellet and supernatant material, have been found as well (7, 8). In these and in other organelles respectively, obtained from centrifugation of the postmito- the only identified molecules with the potential of storing chondrial supernatant at 140,000 x g for 30 min. genetic information are DNAs. Radiolabeling the Products of Mitochondrial RNA (mtRNA) Another interesting feature of plant mitochondrial DNA Synthesis. Mitochondria purified from 30 g of tissue, without (mtDNA) is that variation in it can result in the phenotype of nuclease treatment, were preincubated for 10 min in 250 ,ul of male sterility. In maize there are four distinct cytoplasmic 60 mM mannitol/3 mg of bovine serum albumin per ml/20 and hence mitochondrial types, S, C, T, and N; S, C, and T mM Tris phosphate (pH 7.3)/10 mM potassium phosphate/ can confer male sterility when present in specific nuclear 150 mM KCl/10 mM MgC12/1 mM EGTA/5 mM sodium genetic backgrounds, whereas N cannot (9). We have used an succinate/5 mM phosphoenolpyruvate/30 Ag of pyruvate The publication costs of this article were defrayed in part by page charge Abbreviations: mtRNA, mitochondrial RNA; bp, base pair(s); Tes, payment. This article must therefore be hereby marked "advertisement" N-[tris(hydroxymethyl)methyl]aminoethanesulfonic acid; ds RNA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. double-stranded RNA. 5175 Downloaded by guest on September 27, 2021 5176 Genetics: Finnegan and Brown Proc. Natl. Acad Sci. USA 83 (1986) kinase per ml/2.5 mM ATP/0.3 mM CTP/0.3 mM GTP/0.1 buffer before eluting the double-stranded RNA (ds RNA) mM UTP. Other additions were made as indicated in the with ethanol-free wash buffer. The aurintricarboxylic acid figure legends. The products of RNA synthesis were labeled was removed by passage of RNA samples through a G-25 by adding [a-32P]UTP to a final specific activity of2 Ci/mmol Sepharose column (12). and incubating at 230C with agitation for 60-90 min. RNA for S1 Nuclease Digestion. RNA dissolved in 50 ,ul of 280 mM use as a hybridization probe was labeled in a reaction mixture NaCl/50 mM NaOAc, pH 4.6/4.5 mM ZnSO4/20 gg of containing 2 ,M UTP at 3000 Ci/mmol. The RNA synthesis heat-denatured salmon sperm DNA per ml was digested with reaction was terminated by collecting the mitochondria in a 2000 units of S1 nuclease per ml at 42°C. The digestion microcentrifuge for 5 min and lysing immediately as de- products were extracted with phenol and precipitated with scribed below. ethanol prior to electrophoretic analysis as above. Purification of mtRNA. Mitochondria were resuspended RNA Transfer and Hybridization. After extensive washing and lysed in 50 mM Tes, pH 7.2/10 mM EDTA/0.2% diethyl of the agarose/urea gels with water, RNA was transferred to pyrocarbonate/2% NaDodSO4. One-half volume of redis- a GeneScreen membrane by overnight capillary blotting with tilled phenol equilibrated with 0.1 M Tes (pH 7.2) was quickly 0.3 M NaCl/0.03 M sodium citrate, as recommended by the added and the lysate was extracted, with shaking, 20 min at manufacturer. The membranes were prehybridized for 20 hr 230C. One-half volume of chloroform/isoamyl alcohol (24:1) at 42°C in 50% formamide/0.2% polyvinylpyrrolidone/0.2% was added prior to separation of the phases. The aqueous bovine serum albumin/0.2% Ficoll/50 mM Tris-HCl, pH phase was reextracted twice in this manner before precipi- 7.5/1.0 M NaCl/0.1% sodium pyrophosphate/1% NaDod- tation of the RNA with ethanol. S04/10% dextran sulfate/100 ,ug of heat-denatured salmon Electrophoretic Analysis of RNA. RNA was dissolved in sperm DNA per ml. Hybridizations, with heat-denatured electrophoresis buffer (45 mM Tris/45 mM boric acid/2 mM radiolabeled mtRNA added as probe, were done under the EDTA)/80% deionized formamide, heated 2 min at 70°C, same conditions for 24 hr. The membranes were washed quickly chilled, and loaded onto a vertical 1% agarose slab gel twice for 10 min with wash buffer (0.3 M NaCl/0.03 M sodium containing 2x concentrated electrophoresis buffer/5 M urea. citrate) at 23°C, twice for 30 min with wash buffer/1% Electrophoresis was carried out at 7 V/cm for -4 hr. RNAs NaDodSO4 at 65°C, and twice for 30 min with 0.1 x wash were visualized by ethidium bromide staining after removal buffer at 23°C before autoradiography. of urea by extensive washing with water. Gels containing radiolabeled samples were treated with 10% acetic acid and dried by suction prior to autoradiography. Escherichia coli RESULTS 23S [2.9 kilobases (kb)] and 16S (1.5 kb) RNAs and rabbit RNA Synthesis by Isolated Maize Mitochondria.