Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.

中文翻译：

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探索与植物叶绿体中编码Photosystem II的D1反应中心蛋白的mRNA相关的蛋白质组。

Photosystem II的D1反应中心蛋白的合成可根据环境和发育线索动态调节。在叶绿体中，许多这种调节发生在转录后水平，但是负责的蛋白质在很大程度上是未知的。为了发现影响psbA表达的蛋白质，我们通过以下方法鉴定了与玉米psbA mRNA相关的蛋白质：（i）叶片组织的甲醛交联，然后反义捕获psbA mRNA的寡核苷酸；（ii）与HCF173进行免疫共沉淀，HCF173是一种已知的结合psbA mRNA的psbA翻译激活剂。S1域蛋白SRRP1和两个RNA识别基元（RRM）域蛋白CP33C和CP33B都富含两种方法。直系同源蛋白质也属于拟南芥先前研究中的富集蛋白质组，该研究采用了设计RNA结合蛋白作为psbA RNA亲和标签。我们在这里显示CP33B在体内与psbA mRNA结合，如先前对CP33C和SRRP1所示。缺乏CP33B和/或CP33C的突变体的免疫印迹，脉冲标记和核糖体谱分析分析在某些条件下检测到D1蛋白水平有所下降，但psbA RNA丰度或翻译没有变化。但是，类似的实验表明，SRRP1在黑暗中抑制psbA核糖体缔合，抑制ycf1核糖体缔合，并促进ndhC mRNA的积累。由于已知SRRP1具有RNA伴侣活性，因此我们假设SRRP1通过调节RNA结构介导了这些作用。