Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis[OPEN]

Experimental evidence shows that plant RNA editing factors bind RNA via contiguous modular one-motif:one-nucleotide interactions and that sequence recognition can be altered using simple rules. RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.

[1]  Shimpei Hayashi,et al.  Elucidation of the RNA Recognition Code for Pentatricopeptide Repeat Proteins Involved in Organelle RNA Editing in Plants , 2013, PloS one.

[2]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[3]  Sandra K. Tanz,et al.  A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites[W][OA] , 2009, The Plant Cell Online.

[4]  I. Small,et al.  The evolution of RNA editing and pentatricopeptide repeat genes. , 2011, The New phytologist.

[5]  Charles S. Bond,et al.  A Combinatorial Amino Acid Code for RNA Recognition by Pentatricopeptide Repeat Proteins , 2012, PLoS genetics.

[6]  Ian Small,et al.  Plant RNA editing , 2010, RNA biology.

[7]  A. Brennicke,et al.  In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase* , 2003, Journal of Biological Chemistry.

[8]  Knut Graichen,et al.  Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors , 2013, PloS one.

[9]  Sandra K. Tanz,et al.  The pentatricopeptide repeat protein OTP82 is required for RNA editing of plastid ndhB and ndhG transcripts. , 2009, The Plant journal : for cell and molecular biology.

[10]  Frédérique Bitton,et al.  Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis , 2004, The Plant Cell Online.

[11]  A. Barkan,et al.  Pentatricopeptide repeat proteins in plants. , 2014, Annual review of plant biology.

[12]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[13]  P. León,et al.  CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts. , 2008, The Plant journal : for cell and molecular biology.

[14]  Axel Brennicke,et al.  RNA editing in plants and its evolution. , 2013, Annual review of genetics.

[15]  T. Shikanai,et al.  Mutagenesis of Individual Pentatricopeptide Repeat Motifs Affects RNA Binding Activity and Reveals Functional Partitioning of Arabidopsis PROTON GRADIENT REGULATION3[C][W] , 2013, The Plant Cell.

[16]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[17]  Y. Liu,et al.  Structural basis for the modular recognition of single-stranded RNA by PPR proteins , 2013, Nature.

[18]  Y. Niwa,et al.  Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. , 2007, Journal of bioscience and bioengineering.

[19]  Ian Small,et al.  Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing. , 2014, The Plant journal : for cell and molecular biology.

[20]  Ian Small,et al.  The potential for manipulating RNA with pentatricopeptide repeat proteins. , 2014, The Plant journal : for cell and molecular biology.

[21]  Stephen J Dolgner,et al.  Understanding and engineering RNA sequence specificity of PUF proteins. , 2009, Current opinion in structural biology.

[22]  Ian Small,et al.  A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype. , 2013, The Plant journal : for cell and molecular biology.

[23]  Chen Chen,et al.  Structural basis for RNA recognition by a dimeric PPR-protein complex , 2013, Nature Structural &Molecular Biology.

[24]  T. Shikanai,et al.  A pentatricopeptide repeat protein acts as a site-specificity factor at multiple RNA editing sites with unrelated cis-acting elements in plastids , 2012, Nucleic acids research.