The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Benjamin Lenzen,Dario Leister,Uwe Ohler,Marie-Kristin Lehniger,Christian Schmitz-Linneweber,Ayako Okuzaki,Jose M Muino,Mathias Labs,Thilo Rühle

doi:10.3390/ijms21165633

Abstract

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

Highlights

Chloroplasts contain genetic information that is essential for photosynthesis
Previous studies using microarrays for the detection of RNA targets of CP31A were not quantitative, amongst other reasons because probes used to detect the different RNAs differed in size and sequence composition and were unable to differentiate between the two strands of a gene [19]
Our data lead us to propose that CP31A associates with all ndh mRNAs and that it prefers ndh mRNAs over any other transcripts

Summary

Introduction

Chloroplasts contain genetic information that is essential for photosynthesis. The expression of this information is realized by a unique mixture of ancestral bacterial and derived eukaryotic features [1]. Chloroplast gene expression can be regulated on the transcriptional level [2,3], but post-transcriptional processes are of at least equal importance [4,5,6]. The abundance of chloroplast mRNAs changes in response to external triggers and functionally related RNAs can be combined into larger groups of transcripts [7,8]. Sigma-factors required for the specificity of the plastid-encoded RNA polymerase have been demonstrated to co-regulate functional classes of chloroplast genes at the level of transcription [9,10]. How co-regulation occurs on the post-transcriptional level, remains unknown

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Conclusion