Quantitative analysis of motifs contributing to the interaction between PLS‐subfamily members and their target RNA sequences in plastid RNA editing

Abstract

SUMMARYIn plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. Target cytidines are specifically recognized by pentatricopeptide repeat (PPR) proteins of the PLS subfamily, which have additional C-terminal E or E-DYW motifs. Recent in silico analysis proposed a model for site recognition by PLS-subfamily PPR proteins, with a correspondence of one PPR motif to one nucleotide, and with the C-terminal last S motif aligning with the nucleotide at position -4 with respect to the editing site. Here, we present quantitative biochemical data on site recognition by four PLS-subfamily proteins: CRR28 and OTP85 are DYW-class members, whereas CRR21 and OTP80 are E-class members. The minimal RNA segments required for high-affinity binding by these PPR proteins were experimentally determined. The results were generally consistent with the in silico-based model; however, we clarified that several PPR motifs, including the C-terminal L2 and S motifs of CRR21 and OTP80, are dispensable for the RNA binding, suggesting distinct contributions of each PPR motif to site recognition. We also demonstrate that the DYW motif interacts with the target C and its 5 0 proximal region (from À3 to 0), whereas the E motif is not involved in binding.

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

“…The most detailed analysis of RNA binding by PLS-class proteins delimited the minimum binding sites for the editing factors CRR28, OTP85, CRR21, and OTP80 (Okuda et al, 2014). This analysis showed that each of these factors binds an RNA sequence containing approximately as many nucleotides as the factor contains PPR motifs, evidence in favor of one motif:one nucleotide binding.…”

“…Position 6 appears to be important for distinguishing between purines (A, G) and pyrimidines (U, C), while position 19 directs recognition of amino (A, C) or keto (G, U) groups (Yagi et al, 2013). Most of these correspondences are based on statistical correlations in alignments between PPR binding factors and their RNA targets, but only a few combinations have been confirmed experimentally, and these only for P motifs, and only in vitro (Barkan et al, 2012;Okuda et al, 2014). Based on statistical correlations, S motifs appear to deviate from the P motif code in some important respects and L motifs differ substantially from P and S motifs at the key position 6 (Barkan et al, 2012;Takenaka et al, 2013a).…”

RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering

“…Members of the PLS subfamily of pentatricopeptide repeat (PPR) motif-containing proteins are trans-factors that specifically recognize ciselements (Kotera et al, 2005;Zehrmann et al, 2009). The recognition code that specifies how PPR proteins interact with cis-elements was proposed recently based on in silico analysis and interactions were confirmed by RNA-protein binding assays (Barkan et al, 2012;Takenaka et al, 2013;Yin et al, 2013;Okuda et al, 2014). Many editing PPR proteins contain a C-terminal DYW domain (Lurin et al, 2004).…”

PPR-SMR protein SOT1 has RNA endonuclease activity

“…The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19).…”