Proceedings of the National Academy of Sciences, 1987
The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [... more The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] has five genes, designated Rbcs-1, -2, -3A, -3B, and -3C. We have measured the steady-state mRNA levels for each of the five genes in various tomato organs using gene-specific oligonucleotides. All five genes are highly expressed in leaves, and transcripts of two genes, Rbcs-3B and Rbcs-3C, account for approximately equal to 60% of the total leaf transcripts. The relative transcript levels in the stem, in nature fruits, and etiolated seedlings (plants germinated and grown in the dark) correspond to 3.2%, 6.5%, and 4.6%, respectively, of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA level in leaves, and no transcripts have been detected in roots and ripe tomato fruits. Only Rbcs-1 and Rbcs-2 are expressed during the photosynthetically active phase of fruit development. Transcripts from these genes and ...
Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscripti... more Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5′ region of n...
RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in al... more RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5'-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5'-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded...
RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of mos... more RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced the group I intron‐containing tRNALeu gene, trnL‐CAA, from Takakia lepidozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides tRNALeu gene consisted of a 35‐bp 5′ exon, a 469‐bp group I intron and a 50‐bp 3′ exon. The intron was inserted between the first and second position of the tRNALeu anticodon. In general, plastid tRNALeu genes with a group I intron code for a TAA anticodon in most land plants. This strongly suggests that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived from the trnL‐CAA transcripts. We demonstrated that the first nucleotide C of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or unspliced tRNALeu transcripts r...
The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be in... more The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.
The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisub... more The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced...
Pentatricopeptide repeat (PPR) proteins are widespread in eukaryotes and in particular, include s... more Pentatricopeptide repeat (PPR) proteins are widespread in eukaryotes and in particular, include several hundred members in land plants. The majority of PPR proteins are localized in mitochondria and plastids, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional level in gene expression. However, many of their functions remain to be characterized. In contrast to vascular plants, the moss Physcomitrella patens has only 105 PPR genes. This number may represent a minimum set of PPR proteins required for post-transcriptional regulation in plant organelles. Here, we review the overall structure of the P. patens PPR gene family and the current status of the functional characterization of moss PPR proteins.
Proceedings of the National Academy of Sciences, 1987
The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [... more The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] has five genes, designated Rbcs-1, -2, -3A, -3B, and -3C. We have measured the steady-state mRNA levels for each of the five genes in various tomato organs using gene-specific oligonucleotides. All five genes are highly expressed in leaves, and transcripts of two genes, Rbcs-3B and Rbcs-3C, account for approximately equal to 60% of the total leaf transcripts. The relative transcript levels in the stem, in nature fruits, and etiolated seedlings (plants germinated and grown in the dark) correspond to 3.2%, 6.5%, and 4.6%, respectively, of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA level in leaves, and no transcripts have been detected in roots and ripe tomato fruits. Only Rbcs-1 and Rbcs-2 are expressed during the photosynthetically active phase of fruit development. Transcripts from these genes and ...
Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscripti... more Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5′ region of n...
RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in al... more RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5'-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5'-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded...
RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of mos... more RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced the group I intron‐containing tRNALeu gene, trnL‐CAA, from Takakia lepidozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides tRNALeu gene consisted of a 35‐bp 5′ exon, a 469‐bp group I intron and a 50‐bp 3′ exon. The intron was inserted between the first and second position of the tRNALeu anticodon. In general, plastid tRNALeu genes with a group I intron code for a TAA anticodon in most land plants. This strongly suggests that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived from the trnL‐CAA transcripts. We demonstrated that the first nucleotide C of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or unspliced tRNALeu transcripts r...
The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be in... more The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.
The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisub... more The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced...
Pentatricopeptide repeat (PPR) proteins are widespread in eukaryotes and in particular, include s... more Pentatricopeptide repeat (PPR) proteins are widespread in eukaryotes and in particular, include several hundred members in land plants. The majority of PPR proteins are localized in mitochondria and plastids, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional level in gene expression. However, many of their functions remain to be characterized. In contrast to vascular plants, the moss Physcomitrella patens has only 105 PPR genes. This number may represent a minimum set of PPR proteins required for post-transcriptional regulation in plant organelles. Here, we review the overall structure of the P. patens PPR gene family and the current status of the functional characterization of moss PPR proteins.
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