Effects of RNA structure and salt concentration on the affinity and kinetics of interactions between pentatricopeptide repeat proteins and their RNA ligands.
ABSTRACT: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA sequences via modular 1-repeat:1-nucleotide interactions. Binding specificity is dictated, in part, by hydrogen bonds between the amino acids at two positions in each PPR motif and the Watson-Crick face of the aligned nucleobase. There is evidence that PPR-RNA interactions can compete with RNA-RNA interactions in vivo, and that this competition underlies some effects of PPR proteins on gene expression. Conversely, RNA secondary structure can inhibit the binding of a PPR protein to its specific binding site. The parameters that influence whether PPR-RNA or RNA-RNA interactions prevail are unknown. Understanding these parameters will be important for understanding the functions of natural PPR proteins and for the design of engineered PPR proteins for synthetic biology purposes. We addressed this question by analyzing the effects of RNA structures of varying stability and position on the binding of the model protein PPR10 to its atpH RNA ligand. Our results show that even very weak RNA structures (?G° ~ 0 kcal/mol) involving only one nucleotide at either end of the minimal binding site impede PPR10 binding. Analysis of binding kinetics using Surface Plasmon Resonance showed that RNA structures reduce PPR10's on-rate and increase its off-rate. Complexes between the PPR proteins PPR10 and HCF152 and their respective RNA ligands have long half-lives (one hour or more), correlating with their functions as barriers to exonucleolytic RNA decay in vivo. The effects of salt concentration on PPR10-RNA binding kinetics showed that electrostatic interactions play an important role in establishing PPR10-RNA interactions but play a relatively small role in maintaining specific interactions once established.
Project description:Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5'- and 3'-exonucleases, resulting in processed RNAs with PPR10 bound at the 5'- or 3'-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5'- or 3'-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation.
Project description:Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ?18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design.
Project description:Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region (i) blocks both 5'?3' and 3'? 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5'-terminus in conjunction with a generic 5'?3' exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10's ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins.
Project description:Nucleus-encoded ribonucleases and RNA-binding proteins influence chloroplast gene expression through their roles in RNA maturation and stability. One mechanism for mRNA 5' end maturation posits that sequence-specific pentatricopeptide repeat (PPR) proteins define termini by blocking the 5'?3' exonucleolytic activity of ribonuclease J (RNase J). To test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three PPR proteins and RNase J, both individually and jointly, in Nicotiana benthamiana. In accordance with the stability-conferring function of the PPR proteins PPR10, HCF152 and MRL1, accumulation of the cognate RNA species atpH, petB and rbcL was reduced when the PPR-encoding genes were silenced. In contrast, RNase J reduction alone or combined with PPR deficiency resulted in reduced abundance of polycistronic precursor transcripts and mature counterparts, which were replaced by intermediately sized species with heterogeneous 5' ends. We conclude that RNase J deficiency can partially mask the absence of PPR proteins, and that RNase J is capable of processing chloroplast mRNAs up to PPR protein-binding sites. These findings support the hypothesis that RNase J is the major ribonuclease responsible for maturing chloroplast mRNA 5' termini, with RNA-binding proteins acting as barriers to its activity.
Project description:The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an ?-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts ZEA MAYS: PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, ATPH: and PSAJ, HAS BEEN DEMONSTRATED TO FOLLOW: a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10: PSAJ: complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein-RNA contacts than inferred PREVIOUSLY: Here we describe the solution structure of the ZmPPR10: ATPH: complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA-protein interface.
Project description:Pentatricopeptide repeat (PPR) proteins are a large family of helical repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3'-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3'-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.
Project description: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.
Project description:The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and 'ii'(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.
Project description:The pentatricopeptide repeat (PPR) proteins characterized by tandem repeats of a degenerate 35-amino-acid motif function in all aspects of organellar RNA metabolism, many of which are essential for organellar gene expression. In this study, we report the characterization of a fission yeast Schizosaccharomyces pombe PPR protein, Ppr10 and a novel Ppr10-associated protein, designated Mpa1. The ppr10 deletion mutant exhibits growth defects in respiratory media, and is dramatically impaired for viability during the late-stationary phase. Deletion of ppr10 affects the accumulation of specific mitochondrial mRNAs. Furthermore, deletion of ppr10 severely impairs mitochondrial protein synthesis, suggesting that Ppr10 plays a general role in mitochondrial protein synthesis. Ppr10 interacts with Mpa1 in vivo and in vitro and the two proteins colocalize in the mitochondrial matrix. The ppr10 and mpa1 deletion mutants exhibit very similar phenotypes. One of Mpa1's functions is to maintain the normal protein level of Ppr10 protein by protecting it from degradation by the mitochondrial matrix protease Lon1. Our findings suggest that Ppr10 functions as a general mitochondrial translational activator, likely through interaction with mitochondrial mRNAs and mitochondrial translation initiation factor Mti2, and that Ppr10 requires Mpa1 association for stability and function.
Project description:Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5' and 3' RNA termini in these regions overlap by approximately 25 nucleotides. The PPR10-binding sites map precisely to these overlapping sequences, and PPR10 is required specifically for the accumulation of RNAs with these termini. These findings show that PPR10 serves as a barrier to RNA decay from either the 5' or 3' direction and that a bound protein provides an alternative to an RNA hairpin as a barrier to 3' exonucleases. The results imply that protein 'caps' at both 5' and 3' ends can define the termini of chloroplast mRNA segments. These results, together with recent insights into bacterial RNA decay, suggest a unifying model for the biogenesis of chloroplast transcript populations and for the determinants of chloroplast mRNA stability.