Pentatricopeptide repeat (PPR) proteins bind RNA via a mechanism that facilitates the customization of sequence specificity. However, natural PPR proteins have irregular features that limit the degree to which their specificity can be predicted and customized. We demonstrate here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein characterization. We show by RNA coimmunoprecipitation sequencing (RIP-seq) that artificial PPR proteins designed to bind the Arabidopsis (Arabidopsis thaliana) chloroplast psbA mRNA bind with high specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass spectrometry showed the psbA translational activator HCF173 and two RNA binding proteins of unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and CP33C binding sites placed them in different locations on psbA mRNA. These results demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other applications that rely on the ability to target a protein to a specified RNA sequence.

Photosystem II (PSII) in chloroplasts and cyanobacteria contains approximately fifteen core proteins, which organize numerous pigments and prosthetic groups that mediate the light-driven water-splitting activity that drives oxygenic photosynthesis. The PSII reaction center protein D1 is subject to photodamage, whose repair requires degradation of damaged D1 and its replacement with nascent D1. Mechanisms that couple D1 synthesis with PSII assembly and repair are poorly understood. We address this question by using ribosome profiling to analyze the translation of chloroplast mRNAs in maize and Arabidopsis mutants with defects in PSII assembly. We found that OHP1, OHP2, and HCF244, which comprise a recently elucidated complex involved in PSII assembly and repair, are each required for the recruitment of ribosomes to psbA mRNA, which encodes D1. By contrast, HCF136, which acts upstream of the OHP1/OHP2/HCF244 complex during PSII assembly, does not have this effect. The fact that the OHP1/OHP2/HCF244 complex brings D1 into proximity with three proteins with dual roles in PSII assembly and psbA ribosome recruitment suggests that this complex is the hub of a translational autoregulatory mechanism that coordinates D1 synthesis with need for nascent D1 during PSII biogenesis and repair.

An RNA-chaperone-like protein stabilizes 3ʹ processed transcripts of photosynthetic electron transfer B (petB) and stimulates the translation of petD and petA i

The D1 subunit of photosystem II is subject to photooxidative damage. Photodamaged D1 must be replaced with nascent D1 to maintain photosynthesis. In plant chloroplasts, D1 photodamage regulates D1 synthesis by modulating translation initiation on psbA mRNA encoding D1, but the underlying mechanisms are unknown. Analyses of reporter constructs in transplastomic tobacco (Nicotiana tabacum) showed that the psbA translational regulator HCF173 activates via a cis-element in the psbA 5′-UTR. However, the psbA UTRs are not sufficient to program light-regulated translation. Instead, the psbA open reading frame represses translation initiation in cis, and D1 photodamage relieves this repression. HCF173 remains bound to the psbA 5′-UTR in the dark and truncation of HCF173 prevents repression in the dark, implicating HCF173 as a mediator of repression. We propose a model that accounts for these and prior observations, which is informed by structures of the Complex I assembly factor CIA30/NDUFAF1. We posit that D1 photodamage relieves a repressive cotranslational interaction between nascent D1 and HCF173's CIA30 domain, that the photosystem II assembly factor HCF136 promotes this repressive interaction, and that these events toggle HCF173 between activating and repressive conformations on psbA mRNA. These findings elucidate a translational rheostat that optimizes photosynthesis in response to shifting light conditions.

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.