Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein

Abstract

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. mitochondria | plastid | RNA binding protein | RNA processing P entatricopeptide repeat (PPR) proteins are a recently recognized class of RNA-binding proteins that profoundly affect gene expression in mitochondria and chloroplasts (reviewed in 1). PPR proteins are defined by tandem arrays of a degenerate 35-aa repeat that is predicted to adopt a helical hairpin structure (2). A wealth of genetic data documents the importance of PPR proteins for organellar RNA metabolism: Mutations in PPRencoding genes cause defects in the splicing, editing, stabilization, processing, or translation of subsets of organellar RNAs, with downstream defects in respiration, photosynthesis, and organismal development.Despite the abundant genetic data implicating PPR proteins in organellar gene expression, little is known about the mechanisms through which they act. RNA coimmunoprecipitation assays have shown several PPR proteins to be associated with their genetically defined RNA targets in native extracts (3-7), and several PPR proteins have been shown to bind specifically to their native RNA ligands in vitro (5,(8)(9)(10). By extrapolation, it is anticipated that most PPR proteins are sequence-specific RNA-binding proteins. However, PPRs do not resemble known RNA-binding motifs. In fact, they are closely related to a proteinbinding motif, the tetratricopeptide repeat (TPR) (2). TPR tracts bind protein ligands along a surface created by stacked helical repeating units (11,12). Structural modeling (2) and circular dichroism data (9) support the view that PPR tracts adopt a structure that is similar to that of TPR tracts. This structure is unusual for an RNA-binding domain, most of which are globular (13). It is reminiscent, however, of the Puf domain, which binds singlestranded RNA along a surface formed by the sta...

Disruption of a Rice Pentatricopeptide Repeat Protein Causes a Seedling-Specific Albino Phenotype and Its Utilization to Enhance Seed Purity in Hybrid Rice Production

“…Fisk et al (1999) first reported a maize PPR gene, (for CHLOROPLAST RNA PROCESSING1), which was implicated by genetic analysis in processing and translation of plastid pet transcripts. Similar effects on plastid transcripts were subsequently observed in other mutants from Arabidopsis (Hashimoto et al, 2003;Meierhoff et al, 2003;Yamazaki et al, 2004;ChateignerBoutin et al, 2008ChateignerBoutin et al, , 2011Chi et al, 2008;Okuda et al, 2009Okuda et al, , 2010Yu et al, 2009;Johnson et al, 2010), rice (Kazama and Toriyama, 2003;Komori et al, 2004;Gothandam et al, 2005), and maize (Williams and Barkan, 2003;Schmitz-Linneweber et al, 2006;Pfalz et al, 2009;Prikryl et al, 2011). Additional evidence for a role of PPR proteins in regulating organelle gene expression has also come from positional cloning of several cytoplasmic male sterility (CMS) restorer genes from petunia (Petunia hybrida; Bentolila et al, 2002) and radish (Raphanus sativus; Brown et al, 2003;Desloire et al, 2003;Koizuka et al, 2003).…”

“…Attempts to identify the in vivo RNA ligands have only been made for a few PPR proteins (Schmitz-Linneweber and Small, 2008;Okuda et al, 2009Okuda et al, , 2010Pfalz et al, 2009;Yu et al, 2009;Johnson et al, 2010;Chateigner-Boutin et al, 2011;Prikryl et al, 2011). Many of the postulated activities of PPR proteins still lack direct biochemical proof and thus leave open the possibility that their roles are more indirect than suspected and may require additional, unidentified factors.…”

“…Maize PPR4 (ZmPPR4) is a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif and it facilitates transsplicing of chloroplast rps12 premRNA (Schmitz-Linneweber et al, 2006). ZmPPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region stabilizes RNA and activates translation (Pfalz et al, 2009;Prikryl et al, 2011).…”

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

“…The PPR protein PPR10 has analogous activities but acts on different chloroplast mRNAs (17). Based on the mechanisms established for PPR10 (18), we hypothesized that the HCF107 binding site maps to the immediate 5′ end of those RNA isoforms that fail to accumulate in its absence and that bound HCF107 blocks 5′ → 3′ RNA degradation. As a first step toward testing this hypothesis, we determined whether rHCF107 binds with specificity to a 26-nt RNA with a 5′ end that maps 2 nt upstream of the HCF107-dependent psbH 5′ terminus.…”

The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes

“…It was recently shown that PPR binding could induce local RNA structure reorganization to make otherwise hidden binding sites more accessible to other proteins or complexes. 22 The ability of PPR proteins to locally influence RNA secondary or tertiary structures likely explains the large variety of RNA processing steps associated with this class of proteins.…”