Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA-binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA-based regulation occurs with the psbA mRNA, which shows light-dependent translation. Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA. We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B's preference for psbA, suggesting that there are other determinants of specificity in vivo.Plants 2020, 9, 367 2 of 15 of target mRNAs for various cpRNPs [7][8][9]. rRNAs and intron-less tRNAs are not or only weakly bound [6,7,10]. Since the cpRNPs do not co-fractionate with polysomal RNAs [6,8,11], they are mainly attributed a function prior to translation within posttranscriptional processes.Prediction algorithms for subcellular localization and shotgun proteome analysis identified all ten cpRNPs of Arabidopsis in the chloroplast [summarized in 2]. Fluorescence microscopy of GFP fusion proteins confirmed the chloroplast localization [8,[12][13][14]. Within the chloroplasts, the stroma is the main destination of cpRNPs, with small amounts also being associated with thylakoids. This was proven by immunological analyses for the five cpRNPs from tobacco [6].The expression of cpRNPs is regulated by various external and internal signals. Light especially leads to an accumulation of cpRNPs [summarized in 2]. In general, cpRNPs are involved in a variety of posttranscriptional processes, including 3'-end processing of RNAs [15], RNA editing [16,17], RNA splicing [7], and RNA stabilization [7,8,10]. Some of these processes are modulated by cpRNPs in response to environmental cues and several cpRNPs have been implicated in different acclimation and stress responses [2,7,13,18]. Such a multi-level and far-reaching regulation by multiple external and internal stimuli is unknown for most other chloroplast RBPs, including PPR proteins. cpRNPs are thus considered as prime candidates for post-transcriptional regulators of plastid gene expression [19].A particularly interesting case of chloroplast gene regulation is the light-induced translation of psbA, which codes for the D1 protein, the core subunit of photosytem II [20][21][22][23]. D1 is constantly damaged, most pronouncedly by excess light and other unfavourable conditions, i.e., cold. As a consequence, D1 is constantly synthesized for the repair of PSII [24][25][26]. Moreover, regulated D1 synthesis for de novo biogenesis of PSII during cell growth requires additional regulatory levels of psbA mRNA translation. Consistently, a number of proteins have been co-purified with the psbA mRNA in Chlamydomonas, spinach, A...

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.

Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA based regulation occurs with the psbA mRNA, which shows light-dependent translation.Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA.We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with vast majority of the psbA transcript pool. The RNA sequence target motif determined in vitro does not fully explain CP33B's preference for psbA, suggesting that there are other determinants of specificity in vivo.

29Chloroplast gene expression is characterized by a multitude of post-transcriptional events as well as long-30 lived mRNAs. Given that the chloroplast is responsible for expressing core components of the 31 photosynthetic apparatus, it is not surprising that the accumulation and processing of chloroplast RNA is 32 heavily affected by various external and internal cues, including light and temperature changes. A multitude 33 of nuclear-encoded RNA-binding proteins (RBPs) are known to be required for chloroplast RNA 34 metabolism, but we do not yet know how chloroplast RBPs convert abiotic signals into gene expression 35 changes. Previous studies showed that chloroplast ribonucleoprotein 31A (CP31A) is required for the 36 stabilization of multiple chloroplast mRNAs in the cold, and that the phosphorylation of CP31A at various 37 residues within its N-terminal acidic domain (AD) can alter its affinity for RNA in vitro. Here, we 38 demonstrate that CP31A shows increased affinity for a large number of chloroplast RNAs in vivo. Loss of 39 CP31A leads to cold sensitivity, and cold-dependent RNA binding and cold resistance both depend on the 40 AD of CP31A. Together, our analysis establishes the AD of CP31A as a key mediator of cold acclimation 41 of the chloroplast transcriptome. 42