Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts

Abstract

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 0 and 3 0 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 0 or 3 0 direction and that a bound protein provides an alternative to an RNA hairpin as a barrier to 3 0 exonucleases. The results imply that protein 'caps' at both 5 0 and 3 0 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.

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

“…The authors proposed that proteins that stabilize chloroplast mRNAs are in limiting concentrations such that only a fraction of newly synthesized transcripts escape rapid degradation. Several nucleus-encoded proteins that stabilize specific chloroplast RNAs have been discovered in maize and Arabidopsis (Barkan et al, 1994;Felder et al, 2001;Meierhoff et al, 2003;Lezhneva and Meurer, 2004;Yamazaki et al, 2004;Beick et al, 2008;Pfalz et al, 2009;Johnson et al, 2010;Stoppel et al, 2011;Hammani et al, 2012), and there is evidence that the majority of chloroplast mRNAs in angiosperms are stabilized by proteins that block exoribonucleases (Ruwe and Schmitz-Linneweber, 2012;Zhelyazkova et al, 2012a). Our results imply that such proteins may typically be in limiting concentrations in mature chloroplasts in angiosperms, as in C. reinhardtii chloroplasts.…”

“…5A). This raises the possibility that chloroplast DNA deficiency impacts the expression of nuclear genes such as HCF107, HCF152, and PPR10, which are required for the stabilization of processed psbH or psaJ RNA (Felder et al, 2001;Meierhoff et al, 2003;Pfalz et al, 2009;Hammani et al, 2012;Zoschke et al, 2012). Yet another response was observed for the plastid rpoB gene, whose mRNA accumulated to greatly elevated levels in w2-mum1 tissue (Fig.…”

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

“…Detection of upstream RNA cleavage products strongly suggests that the processing mediated by these proteins involves the recruitment of an endoribonuclease and not a block to exonucleolytic trimming initiated at the 5' extremity of precursor mRNAs, as proposed for other PPR proteins in plastids and mitochondria. 56,57 This mechanism is reminiscent of the mode of action of Rf-PPR proteins, such as RF1A in rice. 6 However, 5'-end processing driven by endonucleolytic cleavage is also associated with mitochondria-targeted PPR proteins that are unrelated to the RFL family, ruling out an exclusive role for RFL proteins in this type of transcript processing.…”

APO1 Promotes the Splicing of Chloroplast Group II Introns and Harbors a Plant-Specific Zinc-Dependent RNA Binding Domain

“…To address the possibility that APO1 binds zinc, Zm-APO1 and At-APO1 were analyzed by inductively coupled argon plasma (ICAP) spectroscopy to determine their metal content. As a control, we analyzed the chloroplast RNA binding protein PPR10 (Pfalz et al, 2009), which consists almost entirely of PPR motifs and is not expected to bind metals. All three proteins were expressed as fusions to MBP in E. coli grown in standard medium without metal supplementation.…”

“…The MBP fusion constructs of Zm-APO1 and At-APO1 (and mutants thereof) are described above. The MBP-PPR10 construct was described previously (Pfalz et al, 2009). These were expressed and purified by amylose affinity chromatography followed by size exclusion chromatography on a Superdex 200 column.…”