title: Predictable alteration of sequence recognition by RNA editing factors from Arabidopsis.
abstract: 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.
authors: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, WA 6009, Australia.
authors: Peter
authors: http://orcid.org/0000-0003-0947-2648
authors: P
authors: Kindgren
authors: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, WA 6009, Australia.
authors: Aaron
authors: A
authors: Yap
authors: School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia.
authors: Charles S
authors: http://orcid.org/0000-0002-9584-6783
authors: CS
authors: Bond
authors: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, WA 6009, Australia ian.small@uwa.edu.au.
authors: Ian
authors: http://orcid.org/0000-0001-5300-1216
authors: I
authors: Small
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017393:RNA Editing / Q000235:genetics*; D018749:RNA, Plant / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D029681:Arabidopsis Proteins; C000603135:CLB19 protein, Arabidopsis; C000603134:OTP82 protein, Arabidopsis; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C538752:pentatricopeptide repeat protein, Arabidopsis
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