Rice Genetics Newsletter, Vol. 18

Rgn18

33.	Transcription and editing status of ribosomal protein S2 gene in rice mitochondria
	N. KUBO¹, A. HIRAI² and K. Kadowaki¹ 1)Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, 305-8602 Japan 2)Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan

Mitochondrial genome of higher plants is much larger in size than that from mammals and insects etc., and often contains additional genes. One of striking examples is presence of ribosomal protein genes in plant mitochondria because all of the ribosomal protein genes are lost from other mitochondrial genomes, such as mammals. In addition to gene contents in the mitochondrial genome, plant mitochondrial genes often have unique characters. It was found by mitochondrial genome sequencing analysis that a gene encoding ribosomal protein S2 (rps2) of rice has an extension in its C-terminal region as compared to its counterpart genes from lower plants and bacteria (Fig. 1; Itadani et al. 1994). This gene contains a part of atpA coding region in the C-terminal extension. However, it is unknown whether or not this "chimeric" rps2 gene is functional. In plant mitochondria, post-transcriptional events are often observed. They are cis-splicing, trans-splicing and RNA editing. Especially, RNA editing is observed in almost all of transcripts from functional protein-coding genes (see, Gott and Emesson 2000 for a review). Therefore, analysis of RNA editing event will be a good clue to know the gene integrity. In this study, we analyzed the transcriptional and post-transcriptional events of the rice rps2 gene.

Transcription of the rice rps2 gene was examined by RNA gel blot analysis. A 2.1-kb signal was detected in rice mitochondrial RNA fraction (Fig. 2A). The size of transcript is enough to cover the entire rps2-coding region (1452 nucleotides). The rps2 gene seems to be actively transcribed in rice mitochondria as inferred from the intensity of the rps2-mRNA signal.

In plant mitochondrial RNA editing, some C-nucleotides are edited into U-nucleotides in RNA, resulting in amino acid changes. In order to know occurrence of editing in the rice rps2 gene transcript, RT-PCR was performed. Rice mitochondrial RNA was reverse-transcribed into cDNA after treatment of RNA with RNase-free DNaseI to remove contaminating mitochondrial genomic DNA. The resultant cDNA was amplified by PCR with primers P1 and P2 (locations of primers are shown in Fig. 1). PCR products were cloned into a pBluescript II SK(+) vector and each clone was sequenced. The editing status was analyzed in five independent cDNA clones (Fig. 2B). There were a total of 10 editing sites in the rps2-coding region

analyzed (Fig. 1). All substitutions were C-to-U change and no U-to-C change (so-called reverse editing) was observed. All of the editing sites are suggested to result in amino acid changes. Of these, eight were located in evolutionarily conserved rps2 region and their positions were identical to those found in wheat rps2 gene (Vaitilingom et al. 1998). One site was observed at N-terminal region which is not conserved in evolution. In addition, one RNA editing site was found in the atpA-coding region in the C-terminal extension.

In conclusion, the above results strongly suggest that the rice rps2 gene encodes a functional protein.

References

Gott, J.M., R.B. Emesson, 2000. Functions and mechanisms of RNA editing. Annu. Rev. Genet. 34: 499-531.

Handa, H., N. Kubo, K. Kadowaki, 1998. Genes for the ribosomal S4 protein encoded in higher plant mitochondria are transcribed, edited, and translated. Mol. Gen. Genet. 258: 199-207.

Itadani, H., T. Wakasugi, M. Sugita, M. Sugiura, M. Nakazono, A. Hirai, 1994. Nucleotide sequence of a 28-kbp portion of rice mitochondrial DNA: the existence of many sequences that correspond to parts of mitochondrial genes in intergenic regions. Plant Cell Physiol. 35: 1239-1244.

Vaitilingom, M., M. Stupar, J.-M. Grienenberger, J. M. Gualberto, 1998. A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons. Mol. Gen. Genet. 258: 530-537.