Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.     

Structure of a PLS-class Pentatricopeptide Repeat Protein Provides Insights into Mechanism of RNA Recognition

Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA-binding proteins that form a pervasive family of proteins conserved in yeast, plants, and humans. The plant PPR proteins are grouped mainly into the P and PLS classes. Here, we report the crystal structure of a PLS-class PPR protein from Arabidopsis thaliana called THA8L (THA8-like) at 2.0 Å. THA8L resembles THA8 (thylakoid assembly 8), a protein that is required for the splicing of specific group II introns of genes involved in biogenesis of chloroplast thylakoid membranes. The THA8L structure contains three P-type PPR motifs flanked by one L-type motif and one S-type motif. We identified several putative THA8L-binding sites, enriched with purine sequences, in the group II introns. Importantly, THA8L has strong binding preference for single-stranded RNA over single-stranded DNA or double-stranded RNA. Structural analysis revealed that THA8L contains two extensive patches of positively charged residues next to the residues that are proposed to comprise the RNA-binding codes. Mutations in these two positively charged patches greatly reduced THA8L RNA-binding activity. On the basis of these data, we constructed a model of THA8L-RNA binding that is dependent on two forces: one is the interaction between nucleotide bases and specific amino acids in the PPR motifs (codes), and the other is the interaction between the negatively charged RNA backbone and positively charged residues of PPR motifs. Together, these results further our understanding of the mechanism of PPR protein-RNA interactions.     

Mutagenesis of Individual Pentatricopeptide Repeat Motifs Affects RNA Binding Activity and Reveals Functional Partitioning of Arabidopsis PROTON GRADIENT REGULATION3

Pentatricopeptide repeat (PPR) proteins bind RNA and act in multiple eukaryotic processes, including RNA editing, RNA stability, and translation. Here, we investigated the mechanism underlying the functional versatility of Arabidopsis thaliana PROTON GRADIENT REGULATION3 (PGR3), a chloroplast protein harboring 27 PPR motifs. Previous studies suggested that PGR3 acts in (1) stabilization of photosynthetic electron transport L (petL) operon RNA, (2) translation of petL, and (3) translation of ndhA. We showed here that replacement of the 4th amino acid of the 12th PPR with nonpolar or charged amino acids abolished functions (1) and (2) but not (3) of PGR3 by compromising the function of this specific PPR. This discovery enabled us to knock out the RNA binding ability of individual PPR motifs. Consequently, we showed that the 16 N-terminal PPRs were sufficient for function (1) via sequence-specific RNA binding, whereas the 11 C-terminal motifs were essential for functions (2) and (3) by activating translation. We also clarified that the 14th amino acid of the 12th PPR should be positively charged to make the PPR functionally active. Our finding opens up the possibility of selectively manipulating the functions of PPR proteins.     

The potential for manipulating RNA with pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence‐specific RNA‐binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2‐30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA‐binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR‐based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.     

Examination of the Dimerization States of the Single-stranded RNA Recognition Protein Pentatricopeptide Repeat 10 (PPR10)

Pentatricopeptide repeat (PPR) proteins, particularly abundant in plastids and mitochrondria of angiosperms, include a large number of sequence-specific RNA binding proteins that are involved in diverse aspects of organelle RNA metabolisms. PPR proteins contain multiple tandom repeats, and each repeat can specifically recognize a RNA base through residues 2, 5, and 35 in a modular fashion. The crystal structure of PPR10 from maize chloroplast exhibits dimeric existence both in the absence and presence of the 18-nucleotide psaJ RNA element. However, previous biochemical analysis suggested a monomeric shift of PPR10 upon RNA binding. In this report, we show that the amino-terminal segments of PPR10 determine the dimerization state of PPR10. A single amino acid alteration of cysteine to serine within repeat 10 of PPR10 further drives dimerization of PPR10. The biochemical elucidation of the determinants for PPR10 dimerization may provide an important foundation to understand the working mechanisms of PPR proteins underlying their diverse physiological functions.     

Molecular cloning and characterization of five genes encoding pentatricopeptide repeat proteins from Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The pentatricopeptide repeat (PPR) protein family is one of the largest and most complex families in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. In this study, we identified many genes encoding PPR protein in Upland cotton through an extensive survey of the database of Gossypium hirsutum. Furthermore, we isolated five full-length cDNA of PPR genes from G. hirsutum 0-613-2R which were named GhPPR1–GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPR1–5 contained from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPR1–2 belonged to the PLS subfamily and GhPPR3–5 belonged to the P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All five GhPPR genes were differentially but constitutively expressed in roots, stems, leaves, pollens, and fibers based on the gene expression analysis by real-time quantitative RT-PCR. This study is the first report and analysis of genes encoding PPR proteins in cotton.     

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.     

Pentatricopeptide repeat proteins and their emerging roles in plants.

Several protein families with tandem repeat motifs play a very important role in plant development and defense. The pentatricopeptide repeat (PPR) protein family, one of the largest families, is the most perplexing one in plants. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. PPR motifs are degenerate motifs, each with 35-amino-acid sequences and are present in tandem arrays of 2-27 repeats per protein. Although PPR proteins are found in other eukaryotes, their large number is probably required in plants to meet the specific needs of organellar gene expression. The repeats of PPR proteins form a superhelical structure to bind a specific ligand, probably a single-stranded RNA molecule, and modulate its expression. Functional studies on different PPR proteins have revealed their role in organellar RNA processing, fertility restoration in CMS plants, embryogenesis, and plant development. Functional genomic techniques can help identify the diverse roles of the PPR family of proteins in nucleus-organelle interaction and in plant development.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.