A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: common use of the same mitochondrial targeting signal for different proteins - PubMed (original) (raw)

A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: common use of the same mitochondrial targeting signal for different proteins

N Kubo et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The rice mitochondrial genome has a sequence homologous to the gene for ribosomal protein S14 (rps14), but the coding sequence is interrupted by internal stop codons. A functional rps14 gene was isolated from the rice nuclear genome, suggesting a gene-transfer event from the mitochondrion to the nucleus. The nuclear rps14 gene encodes a long N-terminal extension showing significant similarity to a part of mitochondrial succinate dehydrogenase subunit B (SDHB) protein from human and a malarial parasite (Plasmodium falciparum). Isolation of a functional rice sdhB cDNA and subsequent sequence comparison to the nuclear rps14 indicate that the 5' portions of the two cDNAs are identical. The sdhB genomic sequence shows that the SDHB-coding region is divided into two exons. Surprisingly, the RPS14-coding region is located between the two exons. DNA gel blot analysis indicates that both sdhB and rps14 are present at a single locus in the rice nucleus. These findings strongly suggest that the two gene transcripts result from a single mRNA precursor by alternative splicing. Protein blot analysis shows that the size of the mature RPS14 is 16.5 kDa, suggesting removal of the N-terminal 22.6-kDa peptide region. Considering that the rice mitochondrial genome lacks the sdhB gene but contains the rps14-related sequence, transfer of the sdhB gene seems to have occurred before the transfer of the rps14 gene. The migration of the mitochondrial rps14 sequence into the already existing sdhB gene could bestow the capacity for nuclear expression and mitochondrial targeting.

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Figures

Figure 1

Figure 1

(A) cDNA and deduced amino acid sequence of the rice nuclear-encoded rps14 gene. The RPS14-homologous region is indicated by a bent arrow. A region homologous to SDHB is underlined. The intron site is marked by an arrowhead. (B) Amino acid sequence comparison of RPS14. Deduced amino acid sequence of rice nuclear rps14 gene (this study) is aligned with amino acid sequences of mitochondrial rps14 genes from rice (this study), broadbean (17), Oenothera (18), rapeseed (19), and liverwort (3). Amino acid residues identical to rice nuclear RPS14 are indicated by dots. Gaps are shown by dashes. An asterisk represents a translational stop codon that truncates an ORF of rice mitochondrial rps14 sequence. Frameshift in the _rps14_-coding region of rice mitochondria was not considered here.

Figure 2

Figure 2

Alignment of deduced amino acid sequences of sdhB genes from rice (this study), human (24, 25), malarial parasite (P. falciparum; DDBJ accession no. D86574), and nuclear rps14 from rice (this study). Amino acid residues identical to the rice SDHB are highlighted by reverse contrast. The three thick bars above the sequence represent three cysteine-rich clusters, which are highly conserved among sdhB genes. An RPS14-homologous region in the rice rps14 gene is indicated by a bent arrow. Arrowheads indicate the intron position of rice sdhB and rps14 genes.

Figure 3

Figure 3

DNA and RNA gel blot analyses of sdhB and nuclear rps14 genes in rice. (A) Schematic representation of sdhB and nuclear rps14 genes. The RPS14-homologous region is shown by a black box. _sdhB_-related regions are shown by hatched boxes. Thick lines indicate the DNA probes used for DNA and RNA gel blot analyses. (B) Rice total DNA (total) and mitochondrial DNA (mt) were digested with _Eco_RI (E) or _Xba_I (X). (Right; probe 3), the bands derived from nuclear DNA are indicated by arrowheads. A molecular length standard is shown at left. (C) Rice poly(A)+ RNA and mitochondrial RNA are represented by (A+) and (mt), respectively. The size of 25S and 17S ribosomal RNAs are indicated at left. The sizes of the transcripts are shown in the figure. A possible primary transcript is indicated by an arrowhead.

Figure 4

Figure 4

Schematic representation of rice nuclear rps14 and sdhB genes. Boxes and thin lines represent protein coding sequence and nontranslated regions, respectively. The box with horizontal stripes represents the rpl5 gene. _rps14_-homologous regions are shown by black boxes. _sdhB_-related regions are shown by hatched boxes. Putative mitochondrial targeting signals of sdhB genes from rice and human are indicated by boxes with both horizontal and vertical lines and the box with vertical stripes, respectively.

Figure 5

Figure 5

Protein blot analysis of rice nuclear RPS14 and SDHB proteins. (A; positive control) Total E. coli protein in which GST–RPS14 fusion protein was overexpressed. (B–D; rice mitochondrial protein) The sera used in the protein analysis and the sizes of the peptides are indicated.

Figure 6

Figure 6

Model for the gene transfer of sdhB and rps14. Rice nucleus and mitochondrion are shown by a square and an enclosure, respectively. Exons and introns are represented by boxes and broken lines, respectively. RPS14-homologous regions are shown by black boxes. _sdhB_-related regions are shown by hatched boxes. Black and hatched circles represent the products of rps14 and sdhB genes, respectively. Other symbols correspond to those in Fig. 4.

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References

    1. Gray M W. Int Rev Cytol. 1992;141:233–357. - PubMed
    1. Brennicke A, Grohmann L, Hiesel R, Knoop V, Schuster W. FEBS Lett. 1993;325:140–145. - PubMed
    1. Oda K, Yamato K, Ohta E, Nakamura Y, Takemura M, Nozato N, Akashi K, Kanegae T, Ogura Y, Kohchi T, et al. J Mol Biol. 1992;223:1–7. - PubMed
    1. Unseld M, Marienfeld J R, Brandt P, Brennicke A. Nat Genet. 1997;15:57–61. - PubMed
    1. Nugent J M, Palmer J D. Cell. 1991;66:473–481. - PubMed

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