RNA Immunoprecipitation and Microarray Analysis Show a Chloroplast Pentatricopeptide Repeat Protein to Be Associated with the 5′ Region of mRNAs Whose Translation It Activates

Abstract

Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 59 untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA-protein interactions in both mitochondria and chloroplasts.

Arabidopsis GLUTAMINE-RICH PROTEIN23Is Essential for Early Embryogenesis and Encodes a Novel Nuclear PPR Motif Protein That Interacts with RNA Polymerase II Subunit III

“…In the Arabidopsis genome, >451 PPR genes have been identified and largely annotated with unknown functions (Aubourg et al, 2000;Small and Peeters, 2000;Lurin et al, 2004). Several loss-of-function mutations of the PPR family have been isolated, and they are mostly involved in RNA processing in mitochondria or chloroplasts (Barkan et al, 1994;Lahmy et al, 2000;Hashimoto et al, 2003;Kazama and Toriyama, 2003;Meierhoff et al, 2003;Nakamura et al, 2003;Lurin et al, 2004;Yamazaki et al, 2004;Kotera et al, 2005;Schmitz-Linneweber et al, 2005). Recently, the identification of seven PPR proteins essential for embryogenesis among the predicted EMB gene data set further extended their function in a developmental context (Cushing et al, 2005).…”

“…The role of the PPR motif is still not clear. It would be interesting to know whether the PPR motif itself can bind DNA or RNA, as PPR proteins have been shown to possess DNA or RNA binding activity (Barkan et al, 1994;Ikeda and Gray, 1999;Lahmy et al, 2000;Small and Peeters, 2000;Mancebo et al, 2001;Liu and McKeehan, 2002;Tsuchiya et al, 2002;Meierhoff et al, 2003;Mili and Pinol-Roma, 2003;Nakamura et al, 2003;Williams and Barkan, 2003;Lurin et al, 2004;Schmitz-Linneweber et al, 2005).…”

“…2003; Mili and Pinol-Roma, 2003;Nakamura et al, 2003;Williams and Barkan, 2003;Lurin et al, 2004;Schmitz-Linneweber et al, 2005). A few functions have been identified, such as those that enable the restoration of cytoplasmic male fertility (Bentolila et al, 2002;Brown et al, 2003;Desloire et al, 2003;Kazama and Toriyama, 2003;Koizuka et al, 2003;Laforest et al, 2003;Akagi et al, 2004;Komori et al, 2004;Klein et al, 2005) and organelle biogenesis (Barkan et al, 1994;Fisk et al, 1999;Hashimoto et al, 2003;Yamazaki et al, 2004;Kotera et al, 2005).…”

The Pentatricopeptide Repeat GeneOTP43Is Required fortrans-Splicing of the Mitochondrialnad1Intron 1 inArabidopsis thaliana

“…Most of the few that have been characterized play a role in posttranscriptional processes in organelles (reviewed in Nakamura et al, 2004;Andres et al, 2007). PPR proteins have been implicated in RNA editing (Kotera et al, 2005;Okuda et al, 2007), RNA processing (Meierhoff et al, 2003;Hattori et al, 2007), RNA splicing (Schmitz-Linneweber et al, 2006), and translational activation (Schmitz-Linneweber et al, 2005). During a reverse genetics screen of PPR mutants, we identified one with a defect in splicing of nad1 transcripts.…”

The next generation of microarray research: applications in evolutionary and ecological genomics

“…ChIP-chip can be used to identify DNA target regions of not only transcription factors but also other proteins that interact with genomes, such as matrix proteins and proteins responsible for chromatin remodeling. RNA immunoprecipitation-chip (RIP-chip) can also be used to determine genome-wide RNA protein binding interactions (Gerber et al, 2004;Schmitz-Linneweber et al, 2005). Another important mechanism of transcriptional regulation is through modulation of the epigenetic states of promoters by cytosine methylation.…”