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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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GMS assays with rCRR22 and target RNAs. (A) RNA sequences used as probes are shown. Editing sites of ndhB-7, ndhD-5 and rpoB-3 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rCRR22 and labeled RNAs (ndhB7L, ndhB7S-S3, ndhD5L, ndhD5S-S3, rpoB3L and rpoB3S-S3), as described in the ‘Materials and Methods’ section. (C) Equilibrium Kd of rCRR22 for the ndhB7S-S3 (left), ndhD5S-S3 (middle) and rpoB3S-S3 (right) probes. rCRR22 concentrations and fractions of RNA bound in each lane are plotted. The Kd calculation assumes a 1:1 interaction between the RNA and the protein. The Kd values and each data point are means ± SD of three experiments performed with the same rCRR22 preparation. All of the GMS assays were performed with the same preparation of rCRR22 and within 2 weeks after purification.
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gks164-F5: GMS assays with rCRR22 and target RNAs. (A) RNA sequences used as probes are shown. Editing sites of ndhB-7, ndhD-5 and rpoB-3 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rCRR22 and labeled RNAs (ndhB7L, ndhB7S-S3, ndhD5L, ndhD5S-S3, rpoB3L and rpoB3S-S3), as described in the ‘Materials and Methods’ section. (C) Equilibrium Kd of rCRR22 for the ndhB7S-S3 (left), ndhD5S-S3 (middle) and rpoB3S-S3 (right) probes. rCRR22 concentrations and fractions of RNA bound in each lane are plotted. The Kd calculation assumes a 1:1 interaction between the RNA and the protein. The Kd values and each data point are means ± SD of three experiments performed with the same rCRR22 preparation. All of the GMS assays were performed with the same preparation of rCRR22 and within 2 weeks after purification.

Mentions: As in the case of OTP82, we first selected sufficiently long sequences surrounding the editing site as RNA probes for the GMS assay. Three 51-nt RNAs, ndhB7L, ndhD5L and rpoB3L, cover the −40 to +10 regions surrounding the ndhB-7, ndhD-5 and rpoB-3 sites, respectively (Figure 5A). A retarded band was detected upon increasing the amounts of rCRR22 (Figure 5B). A 300-fold excess of rCRR22, relative to the ndhB7L, ndhD5L and rpoB3L probes, was required for retardation (Figure 5B). To shift all of the free RNA, a 300- to 1000-fold excess of rCRR22 was needed (Figure 5B).Figure 5.


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)

GMS assays with rCRR22 and target RNAs. (A) RNA sequences used as probes are shown. Editing sites of ndhB-7, ndhD-5 and rpoB-3 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rCRR22 and labeled RNAs (ndhB7L, ndhB7S-S3, ndhD5L, ndhD5S-S3, rpoB3L and rpoB3S-S3), as described in the ‘Materials and Methods’ section. (C) Equilibrium Kd of rCRR22 for the ndhB7S-S3 (left), ndhD5S-S3 (middle) and rpoB3S-S3 (right) probes. rCRR22 concentrations and fractions of RNA bound in each lane are plotted. The Kd calculation assumes a 1:1 interaction between the RNA and the protein. The Kd values and each data point are means ± SD of three experiments performed with the same rCRR22 preparation. All of the GMS assays were performed with the same preparation of rCRR22 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-F5: GMS assays with rCRR22 and target RNAs. (A) RNA sequences used as probes are shown. Editing sites of ndhB-7, ndhD-5 and rpoB-3 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rCRR22 and labeled RNAs (ndhB7L, ndhB7S-S3, ndhD5L, ndhD5S-S3, rpoB3L and rpoB3S-S3), as described in the ‘Materials and Methods’ section. (C) Equilibrium Kd of rCRR22 for the ndhB7S-S3 (left), ndhD5S-S3 (middle) and rpoB3S-S3 (right) probes. rCRR22 concentrations and fractions of RNA bound in each lane are plotted. The Kd calculation assumes a 1:1 interaction between the RNA and the protein. The Kd values and each data point are means ± SD of three experiments performed with the same rCRR22 preparation. All of the GMS assays were performed with the same preparation of rCRR22 and within 2 weeks after purification.
Mentions: As in the case of OTP82, we first selected sufficiently long sequences surrounding the editing site as RNA probes for the GMS assay. Three 51-nt RNAs, ndhB7L, ndhD5L and rpoB3L, cover the −40 to +10 regions surrounding the ndhB-7, ndhD-5 and rpoB-3 sites, respectively (Figure 5A). A retarded band was detected upon increasing the amounts of rCRR22 (Figure 5B). A 300-fold excess of rCRR22, relative to the ndhB7L, ndhD5L and rpoB3L probes, was required for retardation (Figure 5B). To shift all of the free RNA, a 300- to 1000-fold excess of rCRR22 was needed (Figure 5B).Figure 5.

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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