Delineation of pentatricopeptide repeat codes for target RNA prediction

Abstract

Members of the pentatricopeptide repeat (PPR) protein family are sequence-specific RNA-binding proteins that play crucial roles in organelle RNA metabolism. Each PPR protein consists of a tandem array of PPR motifs, each of which aligns to one nucleotide of the RNA target. The di-residues in the PPR motif, which are referred to as the PPR codes, determine nucleotide specificity. Numerous PPR codes are distributed among the vast number of PPR motifs, but the correlation between PPR codes and RNA bases is poorly understood, which hinders target RNA prediction and functional investigation of PPR proteins. To address this issue, we developed a modular assembly method for high-throughput construction of designer PPRs, and by using this method, 62 designer PPR proteins containing various PPR codes were assembled. Then, the correlation between these PPR codes and RNA bases was systematically explored and delineated. Based on this correlation, the web server PPRCODE (http://yinlab.hzau.edu.cn/pprcode) was developed. Our study will not only serve as a platform for facilitating target RNA prediction and functional investigation of the large number of PPR family proteins but also provide an alternative strategy for the assembly of custom PPRs that can potentially be used for plant organelle RNA manipulation.

Figure 1.

Modular assembly efficiency was explored using different numbers of monomers. (A) PPR motif conservation analysis and scaffold sequence selection. The sequence conservation analysis was performed with WebLogo (http://weblogo.berkeley.edu/logo.cgi). The most highly conserved residues at each position were VVTYNTLIDGLCKAGKLDEALKLFEEMVEKGIKPD. To facilitate the cloning and hierarchical ligation of PPR repeats, the codon degeneracy of GL was utilized to construct monomers with different sticky ends. The scaffold module was composed of 25 C-terminal residues (LCKAGKLDEALKLFEEMVEKGIKPD) of repeat X and 10 N-terminal residues (VVTYNTLIDG) of repeat X+1. Ligated residues G and L are colored in pink. Residues N and D are colored in light blue and red, respectively. (B) Schematic diagram of monomer ligation. Two to seven PPR repeats were tested by simultaneous ligation. (C) PCR screening of the ligated 2–7-repeat assemblies. The stars indicate bands with the expected molecular weights. The figures are representative of three replicates. (D) Statistical results for the positive rates of the ligated repeats. The clones were verified by DNA sequencing.

Figure 2.

Flowchart of hierarchical ligation and modular assembly for the construction of custom PPRs. Nine individual PCRs with specific primers were performed to obtain the basic assembly units. Each monomer was customized with specific PPR codes, and each PCR product had a unique linker specifying the position of the PCR product in the assembly. After enzymatic digestion with a type II restriction endonuclease, orthogonal overhangs were derived at the junction position using distinct codons to preserve the same amino acids. The unique overhangs facilitated the positioning of each monomer in the ligation product. The fragments were cloned into pPR18 and verified by sequencing during assembly. The final fragments were cloned into a modified pET21b (ND) vector containing NTD and CTD sequences from PPR10 and a whole repeat divided at the GL junction to form a 10-repeat dPPR.

Figure 3.

The RNA-binding landscape of the designer PPR was examined by EMSA. (A) Schematic diagram of the modularly assembled designer PPRs. The constructed designer PPRs consisted of 10 consecutive PPR repeats containing the ‘ND’ code at the 5th and 35th positions, except repeat five and six, in which desired PPR codes were used. Sixty-two designer PPRs were constructed. (B) RNA substrates were used to determine the RNA-binding specificity of the designer PPRs. These RNAs were 5′ FAM labeled. (C) The RNA-binding specificity of designer PPRs was examined by EMSA. The final protein concentration for each sample was 1 μM. The 5th and 35th di-residues above each lane represent a designer PPR protein containing the corresponding PPR code at repeats five and six. The di-residues are arranged by distribution frequency decreasing from left to right, corresponding to Supplementary Figure S1. B: bound. U: unbound. This figure is representative of three replicates.

Figure 4.

The RNA-binding affinity of the designer PPR was examined by ITC. (A) ITC binding curves show the distinct binding affinity of the dPPR against four RNA substrates. The results are exemplified by four known PPR codes. (B) Binding affinity of each designer PPR against four RNA substrates. The figure was constructed using R. The binding affinities are categorized into three levels (strong, medium and weak); ‘Strong,’ shown in dark blue, represents K_d values in the range of 10–100 nM; ‘Medium,’ shown in slate, represents K_d values in the range of 100–1000 nM; ‘Weak,’ shown in seashell, represents K_d values larger than 1 μM. ‘Null,’ shown in Alice blue, represents no detectable binding by ITC.

Figure 5.

RNA target prediction by the PPRCODE web server. (A) RNA target prediction and comparison of a functionally identified PPR, namely, SOT1 from Arabidopsis. (B) RNA target prediction of a functionally investigated PPR with an unidentified RNA sequence, namely, EMP12 from maize. (C) RNA target prediction of PPR proteins with unknown function, namely, GLYMA11G11880.1 from Glycine max and BV7U_180180_ANIA.T1 from Beta vulgaris.