The small PPR protein SPR2 interacts with PPR-SMR1 to facilitate the splicing of introns in maize mitochondria.

Splicing of plant mitochondrial introns is facilitated by numerous nucleus-encoded protein factors. Although some splicing factors have been identified in plants, the mechanism underlying mitochondrial intron splicing remains largely unclear. In this study, we identified a small P-type pentatricopeptide repeat (PPR) protein containing merely four PPR repeats, small PPR protein 2 (SPR2), which is required for the splicing of more than half of the introns in maize (Zea mays) mitochondria. Null mutations of Spr2 severely impair the splicing of 15 out of the 22 mitochondrial Group II introns, resulting in substantially decreased mature transcripts, which abolished the assembly and activity of mitochondrial complex I. Consequently, embryogenesis and endosperm development were arrested in the spr2 mutants. Yeast two-hybrid, luciferase complementation imaging, bimolecular fluorescence complementation, and semi-in vivo pull-down analyses indicated that SPR2 interacts with small MutS-related domain protein PPR-SMR1, both of which are required for the splicing of 13 introns. In addition, SPR2 and/or PPR-SMR1 interact with other splicing factors, including PPR proteins EMPTY PERICARP16, PPR14, and chloroplast RNA splicing and ribosome maturation (CRM) protein Zm-mCSF1, which participate in the splicing of specific intron(s) of the 13 introns. These results prompt us to propose that SPR2/PPR-SMR1 serves as the core component of a splicing complex and possibly exerts the splicing function through a dynamic interaction with specific substrate recognizing PPR proteins in mitochondria.

DEK48 Is Required for RNA Editing at Multiple Mitochondrial Sites and Seed Development in Maize.

In flowering plants, C-to-U RNA editing can be critical to normal functions of mitochondrion-encoded proteins. Mitochondrial C-to-U RNA editing is facilitated by many factors from diverse protein families, of which the pentatricopeptide repeat (PPR) proteins play an important role. Owing to their large number and frequent embryo lethality in mutants, functions of many PPRs remain unknown. In this study, we characterized a mitochondrion-localized DYW-type PPR protein, DEK48, functioning in the C-to-U RNA editing at multiple mitochondrial transcripts in maize. Null mutation of Dek48 severely arrests embryo and endosperm development, causing a defective kernel (dek) phenotype, named dek48. DEK48 loss of function abolishes the C-to-U editing at nad3-185, -215, and nad4-376, -977 sites and decreases the editing at 11 other sites, resulting in the alteration of the corresponding amino acids. Consequently, the absence of editing caused reduced assembly and activity of complex I in dek48. Interestingly, we identified a point mutation in dek48-3 causing a deletion of the Tryptophan (W) residue in the DYW motif that abolishes the editing function. In sum, this study reveals the function of DEK48 in the C-to-U editing in mitochondrial transcripts and seed development in maize, and it demonstrates a critical role of the W residue in the DYW triplet motif of DEK48 for the C-to-U editing function in vivo.

SMALL KERNEL4 is required for mitochondrial cox1 transcript editing and seed development in maize.

In land plants, cytidine-to-uridine (C-to-U) editing of organellar transcripts is an important post-transcriptional process, which is considered to remediate DNA genetic mutations to restore the coding of functional proteins. Pentatricopeptide repeat (PPR) proteins have key roles in C-to-U editing. Owing to its large number, however, the biological functions of many PPR proteins remain to be identified. Through characterizing a small kernel4 (smk4) mutant, here we report the function of Smk4 and its role in maize growth and development. Null mutation of Smk4 slows plant growth and development, causing small plants, delayed flowering time, and small kernels. Cloning revealed that Smk4 encodes a new E-subclass PPR protein, and localization indicated that SMK4 is exclusively localized in mitochondria. Loss of Smk4 function abolishes C-to-U editing at position 1489 of the cytochrome c oxidase1 (cox1) transcript, causing an amino acid change from serine to proline at 497 in Cox1. Cox1 is a core component of mitochondrial complex IV. Indeed, complex IV activity is reduced in the smk4, along with drastically elevated expression of alternative oxidases (AOX). These results indicate that SMK4 functions in the C-to-U editing of cox1-1489, and this editing is crucial for mitochondrial complex IV activity, plant growth, and kernel development in maize.

PPR-SMR1 is required for the splicing of multiple mitochondrial introns, interacts with Zm-mCSF1, and is essential for seed development in maize.

Group II introns are ribozymes that can excise themselves from precursor-RNA transcripts, but plant organellar group II introns have structural deviations that inhibit ribozyme activity. Therefore, splicing of these introns requires the assistance of nuclear- and/or organellar-encoded splicing factors; however, how these splicing factors function remains unclear. In this study, we report the functions and interactions of two splicing factors, PPR-SMR1 and Zm-mCSF1, in intron splicing in maize mitochondria. PPR-SMR1 is a SMR domain-containing pentatricopeptide repeat (PPR) protein and Zm-mCSF1 is a CRM domain-containing protein, and both are targeted to mitochondria. Loss-of-function mutations in each of them severely arrests embryogenesis and endosperm development in maize. Functional analyses indicate that PPR-SMR1 and Zm-mCSF1 are required for the splicing of most mitochondrial group II introns. Among them, nad2-intron 2 and 3, and nad5-intron 1 are PPR-SMR1/Zm-mCSF1-dependent introns. Protein interaction assays suggest that PPR-SMR1 can interact with Zm-mCSF1 through its N-terminus, and that Zm-mCSF1 is self-interacting. Our findings suggest that PPR-SMR1, a novel splicing factor, acts in the splicing of multiple group II introns in maize mitochondria, and the protein-protein interaction between it and Zm-mCSF1 might allow the formation of large macromolecular splicing complexes.