HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

Abstract

The psbB-psbT-psbH-petB-petD operon of higher plant chloroplasts is a heterogeneously composed transcriptional unit that undergoes complex RNA processing events until the mature oligocistronic RNAs are formed. To identify the nucleusencoded factors required for the processing and expression of psbB-psbT-psbH-petB-petD transcripts, we performed mutational analysis using Arabidopsis. The allelic nuclear mutants hcf152-1 and hcf152-2 were identified that are affected specifically in the accumulation of the plastidial cytochrome b 6 f complex. In both mutants, reduced amounts of spliced petB RNAs (encoding the cytochrome b 6 subunit) were detected, thus explaining the observed protein deficiencies. Additionally, mutant hcf152-1 is affected in the accumulation of transcripts cleaved between the genes psbH and petB . As a result of a close T-DNA insertion, the HCF152 gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF152 encodes a pentatricopeptide repeat (PPR) protein with 12 putative PPR motifs that is located inside the chloroplast. The protein shows a significant structural, but not primary, sequence similarity to the maize protein CRP1, which is involved in the processing and translation of the chloroplast petD and petA RNAs. In addition, we found that HCF152 is an RNA binding protein that binds certain areas of the petB transcript. The protein possibly exists in the chloroplast as a homodimer and is not associated with other proteins to form a high molecular mass complex.

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 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.…”

Chloroplast RNA-binding proteins

“…UV-crosslinking assays with various RNA probes from the psbB-psbT-psbH-petBpetD operon revealed a high affinity binding of recombinant HCF152 to distinct RNA regions covering either the psbH-petB intergenic region or the exon-intron boundaries of petB transcripts. These data suggest that HCF152 is directly involved in the maturation of petB mRNA (Meierhoff et al 2003).…”

“…To date, 16 nuclear loci have been isolated, encoding proteins involved in a variety of post-transcriptional steps of chloroplast gene expression. Although some of these factors have been shown to be part of high-molecular-weight complexes which contain their target RNAs, a direct interaction with plastid RNA probes has recently been documented for just one protein, called HCF152, from Arabidopsis thaliana (Meierhoff et al 2003). HCF152 is involved in the processing of transcripts from the chloroplast psbB operon and contains a so-called pentatricopeptide repeat (PPR) motif, which has been hypothesized to form a RNA-binding domain (Lahmy et al 2000;Small and Peeters 2000).…”