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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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Competition assays demonstrating sequence-specific interactions between rOTP82 and the target RNAs. (A) Two RNAs used as competitors (ndhD1 and ndhD2) are shown. Editing sites of ndhD-1 and ndhD-2 are indicated in bold and are marked with arrowheads. (B) Binding reactions included radioactive ndhB9S or ndhG1S RNA with a 1-, 10- or 100-fold molar excess of the non-radioactive RNA indicated above each panel. The concentration of rOTP82 was held constant at 56 nM. (C) GMS assays were performed with the indicated concentrations of rOTP82 and radioactive RNAs (ndhD1 and ndhD2). All of the competition and GMS assays were performed with the same preparation of rOTP82 as used in Figure 3 and within 2 weeks after purification.
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gks164-F4: Competition assays demonstrating sequence-specific interactions between rOTP82 and the target RNAs. (A) Two RNAs used as competitors (ndhD1 and ndhD2) are shown. Editing sites of ndhD-1 and ndhD-2 are indicated in bold and are marked with arrowheads. (B) Binding reactions included radioactive ndhB9S or ndhG1S RNA with a 1-, 10- or 100-fold molar excess of the non-radioactive RNA indicated above each panel. The concentration of rOTP82 was held constant at 56 nM. (C) GMS assays were performed with the indicated concentrations of rOTP82 and radioactive RNAs (ndhD1 and ndhD2). All of the competition and GMS assays were performed with the same preparation of rOTP82 as used in Figure 3 and within 2 weeks after purification.

Mentions: To analyze whether OTP82 bound to its targets in a sequence-specific manner, we used a set of competitor RNAs to assess interfering effects in the binding of rOTP82 to radioactive RNA probes. As competitors that do not include the OTP82-binding site, we selected two 36-nt RNAs, ndhD1 and ndhD2, which contain the CRR4 binding site and the CRR21 putative binding site, respectively (19,47). They cover the −25 to +10 regions surrounding the ndhD-1 and ndhD2 editing sites, respectively (Figure 4A). The addition of 10 times the amounts of competitor RNAs, ndhB9S and ndhG1S, to the radioactive ndhB9S probe partially blocked retardation, and a 100-fold excess of competitor RNAs completely eliminated retardation (Figure 4B). The addition of the competitor RNAs ndhB9S and ndhG1S to the radioactive ndhG1S probe also interfered with retardation (Figure 4B). In contrast, the addition of a 100-fold excess of the competitor RNAs ndhD1S and ndhD2S did not affect OTP82 binding (Figure 4B). We further confirmed that rOTP82 did not bind to the 32P-labeled ndhD1 and ndhD2 RNAs, even if a 1000-fold excess of rOTP82 was added (Figure 4C). The results of the binding experiments strongly suggested that OTP82 specifically recognized the RNA nucleotides within the −15 to 0 region surrounding the editing sites. Taken together with the genetic evidence indicating that OTP82 is required for RNA editing of ndhB-9 and ndhG-1(41), these findings indicate that OTP82 acts as a site-specificity factor at two sites in plastids.Figure 4.


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)

Competition assays demonstrating sequence-specific interactions between rOTP82 and the target RNAs. (A) Two RNAs used as competitors (ndhD1 and ndhD2) are shown. Editing sites of ndhD-1 and ndhD-2 are indicated in bold and are marked with arrowheads. (B) Binding reactions included radioactive ndhB9S or ndhG1S RNA with a 1-, 10- or 100-fold molar excess of the non-radioactive RNA indicated above each panel. The concentration of rOTP82 was held constant at 56 nM. (C) GMS assays were performed with the indicated concentrations of rOTP82 and radioactive RNAs (ndhD1 and ndhD2). All of the competition and GMS assays were performed with the same preparation of rOTP82 as used in Figure 3 and within 2 weeks after purification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367199&req=5

gks164-F4: Competition assays demonstrating sequence-specific interactions between rOTP82 and the target RNAs. (A) Two RNAs used as competitors (ndhD1 and ndhD2) are shown. Editing sites of ndhD-1 and ndhD-2 are indicated in bold and are marked with arrowheads. (B) Binding reactions included radioactive ndhB9S or ndhG1S RNA with a 1-, 10- or 100-fold molar excess of the non-radioactive RNA indicated above each panel. The concentration of rOTP82 was held constant at 56 nM. (C) GMS assays were performed with the indicated concentrations of rOTP82 and radioactive RNAs (ndhD1 and ndhD2). All of the competition and GMS assays were performed with the same preparation of rOTP82 as used in Figure 3 and within 2 weeks after purification.
Mentions: To analyze whether OTP82 bound to its targets in a sequence-specific manner, we used a set of competitor RNAs to assess interfering effects in the binding of rOTP82 to radioactive RNA probes. As competitors that do not include the OTP82-binding site, we selected two 36-nt RNAs, ndhD1 and ndhD2, which contain the CRR4 binding site and the CRR21 putative binding site, respectively (19,47). They cover the −25 to +10 regions surrounding the ndhD-1 and ndhD2 editing sites, respectively (Figure 4A). The addition of 10 times the amounts of competitor RNAs, ndhB9S and ndhG1S, to the radioactive ndhB9S probe partially blocked retardation, and a 100-fold excess of competitor RNAs completely eliminated retardation (Figure 4B). The addition of the competitor RNAs ndhB9S and ndhG1S to the radioactive ndhG1S probe also interfered with retardation (Figure 4B). In contrast, the addition of a 100-fold excess of the competitor RNAs ndhD1S and ndhD2S did not affect OTP82 binding (Figure 4B). We further confirmed that rOTP82 did not bind to the 32P-labeled ndhD1 and ndhD2 RNAs, even if a 1000-fold excess of rOTP82 was added (Figure 4C). The results of the binding experiments strongly suggested that OTP82 specifically recognized the RNA nucleotides within the −15 to 0 region surrounding the editing sites. Taken together with the genetic evidence indicating that OTP82 is required for RNA editing of ndhB-9 and ndhG-1(41), these findings indicate that OTP82 acts as a site-specificity factor at two sites in plastids.Figure 4.

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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