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A pentatricopeptide repeat protein acts as a site-specificity factor at multiple RNA editing sites with unrelated cis-acting elements in plastids.

Okuda K, Shikanai T - Nucleic Acids Res. (2012)

Bottom Line: Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids.In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein.The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Faculty of Science and Engineering, Chuo University, Bunkyo-ku, Tokyo 112-8551, Japan. okudak@kc.chuo-u.ac.jp

ABSTRACT
In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. All of the site-specificity factors of RNA editing identified so far are pentatricopeptide repeat (PPR) proteins. A defect in a specific PPR protein often impairs RNA editing at multiple sites, at which the cis-acting elements are not highly conserved. The molecular mechanism for sharing a single PPR protein over multiple sites is still unclear. We focused here on the PPR proteins OTP82 and CRR22, the putative target elements of which are, respectively, partially and barely conserved. Recombinant OTP82 specifically bound to the -15 to 0 regions of its target sites. Recombinant CRR22 specifically bound to the -20 to 0 regions of the ndhB-7 and ndhD-5 sites and to the -17 to 0 region of the rpoB-3 site. Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids. In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein. The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein.

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Two models to explain the function of a single PPR protein that functions at multiple sites. Alignments of the nucleotide sequences in the region surrounding the editing sites affected in (A) otp82 and (B) crr22 are shown. The alignments include the sequences from −20 to +5 around the edited C (bold), with identical nucleotides shown in shaded boxes. Consensus sequences of the 15 nt immediately upstream of the edited C were identified by bioinformatics analysis (20) and are shown above the target sequences. In the consensus, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are indicated. (C). In the upper model, each cis-acting element is recognized by the indicated PPR protein (medium and dark gray) and a second PPR protein (light gray) functions as a binding partner shared via the formation of a heterodimer at each site. The PPR protein may have additional functions other than RNA recognition, such as providing the DYW motif for the editing reaction. In the lower model, the PPR protein acts as a site-specificity factor at multiple sites.
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gks164-F1: Two models to explain the function of a single PPR protein that functions at multiple sites. Alignments of the nucleotide sequences in the region surrounding the editing sites affected in (A) otp82 and (B) crr22 are shown. The alignments include the sequences from −20 to +5 around the edited C (bold), with identical nucleotides shown in shaded boxes. Consensus sequences of the 15 nt immediately upstream of the edited C were identified by bioinformatics analysis (20) and are shown above the target sequences. In the consensus, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are indicated. (C). In the upper model, each cis-acting element is recognized by the indicated PPR protein (medium and dark gray) and a second PPR protein (light gray) functions as a binding partner shared via the formation of a heterodimer at each site. The PPR protein may have additional functions other than RNA recognition, such as providing the DYW motif for the editing reaction. In the lower model, the PPR protein acts as a site-specificity factor at multiple sites.

Mentions: A defect in a single PPR protein often impairs RNA editing of multiple sites (20–22,27,29). The hypothesis that a site-specificity factors can be shared by multiple sites was originally suggested by the finding that high-level accumulation of RNA including a single editing site decreases editing efficiency at other sites (31,32). Subsequently, this hypothesis was confirmed by using in vitro RNA editing systems with chloroplast extracts (33). In addition, transcripts encompassing two editing sites, ZMrpoB C467 and ZMrps14 C80, competed against each other for editing activity in vitro (34). An analysis of the target sites of five PPR proteins involved in RNA editing of multiple sites revealed an unambiguous 15-nt consensus, providing sufficient specificity to define the edited sites in the plastid transcriptome (20). An in silico search of the Arabidopsis mitochondrial genome revealed that two sites recognized by OTP87 contained sufficient conserved nucleotides to be unambiguously defined as a binding site consensus; moreover, biochemical evidence for the specific binding of OTP87 to multiple editing sites was provided (30). However, the molecular mechanism by which a trans-acting factor is shared by multiple sites is still not fully clear, because the cis-acting elements recognized by a single PPR protein are often not conserved. The most prominent examples are the plastid targets of the PPR protein CRR22: the ndhB-7, ndhD-5 and rpoB-3 sites (35). In contrast to previous reports, in which multiple target sequences recognized by a single PPR protein have been almost identical (36,37) or partially conserved (20,27,38), the sequences surrounding these editing sites appear divergent (Figure 1). It was proposed that the DYW motif could catalyze the editing reaction, because it contains some residues that are conserved in the human C deaminase, APOBEC-1 (7). Since the catalytically active form of APOBEC-1 is an asymmetric homodimer (39), DYW-subclass PPR protein may likewise dimerize. Since different editing sites targeted by the same PPR protein do not share high conservation, it is plausible that the PPR protein plays different roles at these editing sites. In this model, two PPR proteins would be heterodimeric: one would act as a site-specificity factor and the other would function to provide the DYW motif for the editing reaction (Figure 1C). This may be the case for CRR22, because it is required for RNA editing events in which the putative cis-acting elements are not conserved (Figure 1B). Alternatively, CRR22 may recognize all the elements as a genuine site-specificity factor. To answer the question of whether a PPR protein involved in the editing of multiple sites with low levels of conservation binds specifically to its different targets, we selected CRR22. We also analyzed OTP82, which is required for two RNA editing events at sites with putative cis-acting elements that are only partially conserved (Figure 1A).Figure 1.


A pentatricopeptide repeat protein acts as a site-specificity factor at multiple RNA editing sites with unrelated cis-acting elements in plastids.

Okuda K, Shikanai T - Nucleic Acids Res. (2012)

Two models to explain the function of a single PPR protein that functions at multiple sites. Alignments of the nucleotide sequences in the region surrounding the editing sites affected in (A) otp82 and (B) crr22 are shown. The alignments include the sequences from −20 to +5 around the edited C (bold), with identical nucleotides shown in shaded boxes. Consensus sequences of the 15 nt immediately upstream of the edited C were identified by bioinformatics analysis (20) and are shown above the target sequences. In the consensus, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are indicated. (C). In the upper model, each cis-acting element is recognized by the indicated PPR protein (medium and dark gray) and a second PPR protein (light gray) functions as a binding partner shared via the formation of a heterodimer at each site. The PPR protein may have additional functions other than RNA recognition, such as providing the DYW motif for the editing reaction. In the lower model, the PPR protein acts as a site-specificity factor at multiple sites.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367199&req=5

gks164-F1: Two models to explain the function of a single PPR protein that functions at multiple sites. Alignments of the nucleotide sequences in the region surrounding the editing sites affected in (A) otp82 and (B) crr22 are shown. The alignments include the sequences from −20 to +5 around the edited C (bold), with identical nucleotides shown in shaded boxes. Consensus sequences of the 15 nt immediately upstream of the edited C were identified by bioinformatics analysis (20) and are shown above the target sequences. In the consensus, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are indicated. (C). In the upper model, each cis-acting element is recognized by the indicated PPR protein (medium and dark gray) and a second PPR protein (light gray) functions as a binding partner shared via the formation of a heterodimer at each site. The PPR protein may have additional functions other than RNA recognition, such as providing the DYW motif for the editing reaction. In the lower model, the PPR protein acts as a site-specificity factor at multiple sites.
Mentions: A defect in a single PPR protein often impairs RNA editing of multiple sites (20–22,27,29). The hypothesis that a site-specificity factors can be shared by multiple sites was originally suggested by the finding that high-level accumulation of RNA including a single editing site decreases editing efficiency at other sites (31,32). Subsequently, this hypothesis was confirmed by using in vitro RNA editing systems with chloroplast extracts (33). In addition, transcripts encompassing two editing sites, ZMrpoB C467 and ZMrps14 C80, competed against each other for editing activity in vitro (34). An analysis of the target sites of five PPR proteins involved in RNA editing of multiple sites revealed an unambiguous 15-nt consensus, providing sufficient specificity to define the edited sites in the plastid transcriptome (20). An in silico search of the Arabidopsis mitochondrial genome revealed that two sites recognized by OTP87 contained sufficient conserved nucleotides to be unambiguously defined as a binding site consensus; moreover, biochemical evidence for the specific binding of OTP87 to multiple editing sites was provided (30). However, the molecular mechanism by which a trans-acting factor is shared by multiple sites is still not fully clear, because the cis-acting elements recognized by a single PPR protein are often not conserved. The most prominent examples are the plastid targets of the PPR protein CRR22: the ndhB-7, ndhD-5 and rpoB-3 sites (35). In contrast to previous reports, in which multiple target sequences recognized by a single PPR protein have been almost identical (36,37) or partially conserved (20,27,38), the sequences surrounding these editing sites appear divergent (Figure 1). It was proposed that the DYW motif could catalyze the editing reaction, because it contains some residues that are conserved in the human C deaminase, APOBEC-1 (7). Since the catalytically active form of APOBEC-1 is an asymmetric homodimer (39), DYW-subclass PPR protein may likewise dimerize. Since different editing sites targeted by the same PPR protein do not share high conservation, it is plausible that the PPR protein plays different roles at these editing sites. In this model, two PPR proteins would be heterodimeric: one would act as a site-specificity factor and the other would function to provide the DYW motif for the editing reaction (Figure 1C). This may be the case for CRR22, because it is required for RNA editing events in which the putative cis-acting elements are not conserved (Figure 1B). Alternatively, CRR22 may recognize all the elements as a genuine site-specificity factor. To answer the question of whether a PPR protein involved in the editing of multiple sites with low levels of conservation binds specifically to its different targets, we selected CRR22. We also analyzed OTP82, which is required for two RNA editing events at sites with putative cis-acting elements that are only partially conserved (Figure 1A).Figure 1.

Bottom Line: Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids.In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein.The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Faculty of Science and Engineering, Chuo University, Bunkyo-ku, Tokyo 112-8551, Japan. okudak@kc.chuo-u.ac.jp

ABSTRACT
In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. All of the site-specificity factors of RNA editing identified so far are pentatricopeptide repeat (PPR) proteins. A defect in a specific PPR protein often impairs RNA editing at multiple sites, at which the cis-acting elements are not highly conserved. The molecular mechanism for sharing a single PPR protein over multiple sites is still unclear. We focused here on the PPR proteins OTP82 and CRR22, the putative target elements of which are, respectively, partially and barely conserved. Recombinant OTP82 specifically bound to the -15 to 0 regions of its target sites. Recombinant CRR22 specifically bound to the -20 to 0 regions of the ndhB-7 and ndhD-5 sites and to the -17 to 0 region of the rpoB-3 site. Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids. In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein. The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein.

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