Chloroplast ribonucleoprotein CP31A is required for editing and stability of specific chloroplast mRNAs

Abstract

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded, highly abundant, and light-regulated RNA binding proteins. They have been shown to be involved in chloroplast RNA processing and stabilization in vitro and are phylogenetically related to the well-described heterogeneous nuclear ribonucleoproteins (hnRNPs). cpRNPs have been found associated with mRNAs present in chloroplasts and have been regarded as nonspecific stabilizers of chloroplast transcripts. Here, we demonstrate that null mutants of the cpRNP family member CP31A exhibit highly specific and diverse defects in chloroplast RNA metabolism. First, analysis of cp31a and cp31a/cp31b double mutants uncovers that these 2 paralogous genes participate nonredundantly in a combinatorial fashion in processing a subset of chloroplast editing sites in vivo. Second, a genome-wide analysis of chloroplast transcript accumulation in cp31a mutants detected a virtually complete loss of the chloroplast ndhF mRNA and lesser reductions for specific other mRNAs. Fluorescence analyses show that the activity of the NADH dehydrogenase complex, which also includes the NdhF subunit, is defective in cp31a mutants. This indicates that cpRNPs are important in vivo for calibrating the expression levels of specific chloroplast mRNAs and impact chloroplast physiology. Taken together, the specificity and combinatorial aspects of cpRNP functions uncovered suggest that these chloroplast proteins are functional equivalents of nucleocytosolic hnRNPs.Arabidopsis ͉ RNA binding ͉ RNA editing

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…Immunodepletion of CP31 from the editing extract resulted in the inhibition of editing of psbL and ndhB mRNAs. More recently, a null mutant of CP31A, one of the two paralogues found in Arabidopsis, was shown to exhibit multiple specific editing defects in chloroplast transcripts (43). However, Tillich et al (43) also observed that CP31 was responsible for the stability of specific chloroplast mRNAs, because almost no ndhF mRNA could be detected, and other chloroplast mRNAs were also depleted in cp31a mutants.…”

“…The editing defect in cp31a mutant and the cp31a/ cp31b double mutant is much less severe than ones that we observed in the orrm1 mutant; an edited peak was detectable in the electrophoretograms of RT-PCR bulk sequences surrounding the editing sites most affected in cp31a/cp31b mutant (figure S2 in ref. 43). Bulk sequencing is a much less sensitive editing assay than either RNA-seq or PPE.…”

Function of PPR proteins in plastid gene expression

“…In addition to ORRM1, another RRM protein, CP31, was suggested to be involved in RNA editing in plastids. 46,74 The editing machinery in plastids may be more complicated than we previously estimated in the two-component model. 34 In addition to a transfactor, the MORF2/MORF9 heterodimer is also necessary for almost all editing sites in plastids.…”

The PHOTOSYNTHESIS AFFECTED MUTANT68–LIKE Protein Evolved from a PSII Assembly Factor to Mediate Assembly of the Chloroplast NAD(P)H Dehydrogenase Complex in Arabidopsis

“…Then, pulses (0.8 s) of white light (5000 mmol photons m 22 s 21 ) were used to determine the maximum fluorescence (Fm). The transient increase in fluorescence was recorded after a 5-min exposure to actinic light (80 mmol photons m 22 s 21 ; as previously described in Tillich et al, 2009). ETR and NPQ of Arabidopsis leaves and in vivo chlorophyll a fluorescence of whole P. patens plants were recorded using the Imaging PAM (Walz).…”

“…In vivo chlorophyll a fluorescence of Arabidopsis leaves was measured using the pulse amplitude modulation 101/103 instrument (PAM 101/103; Walz) as described (Varotto et al, 2000;Tillich et al, 2009). Plants were dark-adapted for 30 min, and minimal fluorescence (Fo) was measured.…”