The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA

Abstract

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...

The Chloroplast RNA Binding Protein CP29A supportsrbcLexpression during cold acclimation

“…The psbA mRNA was confirmed as a target for CP33B, with no corresponding peak in the CP29A dataset (Figure 1C). In contrast, rbcL mRNA was a target of CP29A but not CP33B, aligning with previous RIP-Chip and dot blot analyses (Figure 1D; Kupsch et al, 2012; Watkins et al, 2019; Teubner et al, 2020).…”

“…Importantly, no sequence-level overlap was observed between significantly enriched peaks in the CP29A and CP33B datasets, indicating distinct specificities. CP33B’s main target was the psbA mRNA, whereas rbcL mRNA was a strong target of CP29A (Figure 1A,B, Table S1), consistent with previous analyses (3, 13, 14).…”

“…We adapted the eCLIP protocol (11,12) for chloroplasts, employing specific antibodies for CP29A and CP33B, another cpRNP family member. The binding pattern of CP33B, revealed through prior RIP-Chip results, differs substantially from CP29A (3,13,14). Analyzing two RBPs with different target specificities in parallel can unveil potential bias and false-positives in the procedure.…”

“…Intron-lacking tRNAs and rRNAs were found to have little or no affinity for cpRNPs (Nakamura et al, 1999). In contrast, cpRNPs were found to associate with various mRNAs and some intron-containing tRNAs (Kupsch et al, 2012; Teubner et al, 2020, 2017; Nakamura et al, 1999). A top-enriched transcript in IPs of CP29A was rbcL (Kupsch et al, 2012).…”

Characterization of the preferred cation cofactors of chloroplast protein kinases in Arabidopsis thaliana

“…At 50 kDa, glyceraldehyde‐3‐phosphate dehydrogenase (involved in the Calvin and Benson cycle) and glutamate‐1‐semialdehyde 2,1‐aminomutase 2 were identified (Table 1 ). Putative substrate candidates at 30 kDa were chloroplasts ribosomal proteins and the RNA‐binding protein CP33B (Table 1 ) known to bind specifically to psbA mRNA [ 50 ]. It is tempting to speculate that an Mn ion‐dependent kinase phosphorylates CP33B thereby exerting a regulatory role in psbA translation.…”