Analysis of RNA Recognition and Binding Characteristics of OsCPPR1 Protein in Rice

doi:10.1016/j.rsci.2023.11.011

Abstract

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 anther. 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, RNA-electrophoretic mobility shift assay experiment demonstrated that the recombinant OsCPPR1 can directly recognize and bind to OsGLK1 mRNA in vitro. 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 co-factors to assist in cleavage, a facet that warrants further exploration in subsequent studies.

Key words: OsCPPR1, RNA recognition and binding, pentatricopeptide repeat, rice

Zheng Shaoyan, Chen Junyu, Li Huatian, Liu Zhenlan, Li Jing, Zhuang Chuxiong. Analysis of RNA Recognition and Binding Characteristics of OsCPPR1 Protein in Rice[J]. Rice Science, 2024, 31(2): 215-225.

Figures/Tables 6

Fig. 1. Sequence alignment of pentatricopeptide repeat (PPR) protein in OsCPPR1. A, Schematic diagram of OsCPPR1 with its 16 PPR motifs. B, Sequence logo generated using PPR sequences of OsCPPR1 reveals relatively conserved amino acid positions within the PPR. C, Multiple sequence alignment of all 16 PPR motifs of OsCPPR1(aa 99-649). The determining residues for RNA recognition at the 5th positions are highlighted by a red box.

Fig. 2. Structure and similarity analysis of OsCPPR1, OsCPPR1(aa 135-end), and OsCPPR1(aa 1-666) proteins. A, Schematic diagram of OsCPPR1, OsCPPR1(aa 135-end), and OsCPPR1(aa 1-666). B, Secondary structure analyses of OsCPPR1, OsCPPR1(aa 135-end), and OsCPPR1(aa 1-666) were conducted using lamdba predict protein (λPP) (https://embed.predictprotein.org). The red box represents the corresponding missing pentatricopeptide repeat (PPR) motif region of OsCPPR1(aa 135-end) and OsCPPR1(aa 1-666). C, All 16 PPR motifs were showed in OsCPPR1 protein of the predicted three-dimensional structure. The PPR motifs were showed by the red arrows, and the black helix represented the PPR motif at the N- and C-terminal. D, Predicted three-dimensional structure (up) and the surface model structure (down) of different truncated OsCPPR1 proteins by Alphafold 2.

Fig. 3. Far-ultraviolet circular dichroism spectra of wild type and different truncated OsCPPR1 proteins.

Fig. 4. Interaction analysis and prediction of OsGLK1-G4 RNA fragment with wild type and truncated OsCPPR1s. A-D, Base interaction prediction of OsGLK1-G4 RNA nucleotide with wild type (OsCPPR1, A) and truncated OsCPPR1s [OsCPPR1m, B; OsCPPR1(aa 135-end), C; OsCPPR1(aa 1-666), D] from the HDOCK server. A1-D1, The interaction analysis details between OsGLK1-G4 RNA nucleotide with wild type (OsCPPR1, A1) and truncated OsCPPR1s [OsCPPR1m, B1; OsCPPR1(aa 135-end), C1; OsCPPR1(aa 1-666), D1] from the HDOCK server.

Fig. 5. RNA-electrophoretic mobility shift assay for binding of wild type and truncated OsCPPR1s to OsGLK1 G4-RNA fragment. RNA fragment G4 from the RIP-PCR (RNA immunoprecipitation quantitative polymerase chain reaction) analysis was end-labeled with biotin, and the same unlabeled RNA fragment (50-fold) was used as a cold competitor. The same concentration RNA fragment G4 was incubated with different truncated OsCPPR1 proteins, respectively.

Fig. 6. Positions of 5′-RNA ligase-mediated rapid amplification of cDNA ends products on OsGLK1 in wild type (WT) and OsCPPR1 overexpression (CPPR1-OE) plants. A, qRT-PCR analysis of OsGLK1 in the flag levels of WT and CPPR1-OE (CPPR1-OE1 and CPPR1-OE2) plants at the flowering stage. Data are Mean ± SD (n = 3). ***, P < 0.001 according to the Student’s t-test. The ACTIN gene was used as an internal standard.B, Summary of cleavage sites on OsGLK1 transcripts in WT and CPPR1-OE (CPPR1-OE1 and CPPR1-OE2) (n > 100 clones).

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