A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.     

A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2. Received: 20 November 1997 / Accepted: 29 January 1998     

Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

Plant mitochondrial rps2 genes code for proteins with a C-terminal extension that is processed

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.     

The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent

Many eukaryotic genomes have experienced ancient whole-genome duplication (WGD) followed by massive gene loss. These eliminations were not random since some gene families were preferentially retained as duplicates. The gene balance hypothesis suggests that those genes with dosage reduction can imbalance their interacting partners or complex, resulting in decreased fitness. In Arabidopsis, the cytoplasmic ribosomal proteins (RP) are encoded by gene families with at least two members. We have focused our study on the two RPS6 genes in an attempt to understand why they have been retained as duplicates. We demonstrate that RPS6 function is vital for the plant. We also show that reducing the level of RPS6 accumulation (in the knock-out rps6a or rps6b single mutants, or in the double heterozygous RPS6A/rps6a,RPS6B/rps6b), confers a slow growth phenotype (haplodeficiency). Importantly, we demonstrate that the functions of two RPS6 genes are redundant and interchangeable. Finally, like in most other described Arabidopsis rp mutants, we observed that a reduced RPS6 level slightly alters the dorsoventral leaf patterning. Our results support the idea that the Arabidopsis RPS6 gene duplicates were evolutionarily retained in order to maintain an expression level necessary to sustain the translational demand of the cell, in agreement with the gene balance hypothesis.     

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.     

Suppression of a defect in the 5'' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.     

Altered mRNA binding activity and decreased translational initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA.

Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.     

RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5' region of mRNAs whose translation it activates

Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 5' untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA-protein interactions in both mitochondria and chloroplasts.