ORIGINAL PAPER M. Pi á T. Morio á H. Urushihara á Y. Tanaka Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA Received: 17 May 1997 / Accepted: 26 August 1997 Abstract In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly dierent from that in mito- chondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mi- tochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary struc- ture of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25 000 ´ g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA. Key words Dictyostelium discoideum á Ribosome á 5S rRNA á Mitochondrial DNA á Small RNA Introduction Mitochondria have their own genome, as well as tran- scription and translation systems resembling those of prokaryotes but dierent from those in the cytoplasm (Gillham 1994). In animal and fungal mitochondria, the genome does not encode 5S rRNA, and 5S rRNA is absent from ribosomes (Attardi and Ojala 1971; Lizardi and Luck 1971; Gillham 1994). Recently, Yoshinari et al. (1994) identi®ed a cytoplasmic 5S rRNA as a soluble component in bovine mitochondria, and suggested that 5S rRNA is present in all vertebrate mitochondria, based on the results of preliminary experiments on rabbit and chicken mitochondria. However, the function of this molecule in mitochondria is unknown. On the other hand, plant mitochondrial (mt) DNAs examined to date have been shown to have a 5S rRNA gene (Spencer et al. 1981; Chao et al. 1983; Morgens et al. 1984; Wissinger et al. 1988; Gray 1992) and the large ribosomal subunit of mung bean mitochondria contains a 5S rRNA in equimolar proportions to large-subunit (LSU) rRNA (Leaver and Harmey 1976). The chloro- phycean alga Prototheca wikerhamii is the ®rst non-plant organism that has been shown to have a 5S rRNA gene in its mtDNA (Wol et al. 1994). Interestingly, the mtDNA of the protozoan Acanthamoeba castellanii has no 5S rRNA gene (Burger et al. 1995), and in the mi- tochondrial genome of the brown alga Pylaiella littoralis the gene is also absent (Fontaine et al. 1995). The pos- sibility that a 5S rRNA gene is present in Chondrus crispus mtDNA (Leblanc et al. 1995) was recently dis- proved (Lang et al. 1996). The cellular slime mold Dictyostelium discoideum has been used as a model system for the study of cell dif- ferentiation, cell movement and cell-cell interactions (Loomis 1982; Kimmel 1988). This organism is also in- teresting from an evolutionary viewpoint, because it shows characteristics of both animals and plants and has both unicellular and multicellular stages in its life cycle. We are currently sequencing and analyzing the 54-kb mtDNA (Fukuhara 1982), mainly with a view to un- derstanding its evolution, and have reported several re- sults which suggest that the genes in, and the organization of, the D. discoideum mt genome show plant-like characteristics (Tanaka et al. 1990; Angata et al. 1995a, b). In the present study, we analyzed a mi- Mol Gen Genet (1998) 257: 124±131 Ó Springer-Verlag 1998 Communicated by D. M. Lonsdale M. Pi á T. Morio á H. Urushihara á Y. Tanaka (&) Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Fax: +81-298-53-6614; e-mail: ytanaka@sakura.cc.tsukuba.ac.jp Y. Tanaka Center for TARA (Tsukuba Advanced Research Alliance), Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan