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Analysis of RNA Recognition and Binding Characteristics of OsCPPR1 Protein

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangdong Laboratory for Lingnan Modern Agriculture, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. # These authors contributed equally to this article
  • Contact: ZHUANG Chuxiong; ZHENG Shaoyan
  • Supported by:

    This research was supported by the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (Grant No. 2022SDZG05), the National Natural Science Foundation of China (Grants No. 32000457, 31871231, and 31921004), the Major Program of Guangdong Basic and Applied Research (Grant No. 2019B030302006), and the Double First-Class Discipline Promotion Project (Grant No. 2021B10564001). We thank Dr. Yu Haopeng, Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK for assistance in the analysis of the structure of OsGLK1 RNA fragments.

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Abstract: Pentatricopeptide repeat (PPR) proteins represent one of the largest protein families in plants and typically localize to organelles like mitochondria and chloroplasts. By contrast, CYTOPLASM-LOCALIZED PPR1 (OsCPPR1) is a cytoplasm-localized PPR protein that can degrade OsGOLDEN-LIKE1 (OsGLK1) mRNA in the tapetum of rice (Oryza sativa) anthers. However, the mechanism by which OsCPPR1 recognizes and binds to OsGLK1 transcripts remains unknown. Through protein structure prediction and macromolecular docking experiments, we observed that distinct PPR motif structures of OsCPPR1 exhibited varying binding efficiencies to OsGLK1 RNA. Moreover, our RNA-EMSA experiment demonstrated that the recombinant CPPR1 can directly recognize and bind to OsGLK1 mRNA in vitro through the RNA-EMSA experiment. This further confirmed that the mutations in the conserved amino acids in each PPR motif resulted in loss of activity, while truncation of OsCPPR1 decreased its binding efficiency. These findings collectively suggest that it may require some cofactor to assist in cleavage, a facet that warrants further exploration in subsequent studies.

Key words: OsCPPR1; RNA recognition and binding, PPR