Hakim Mireau

Ribozymic introns are widely distributed in eubacteria and organelles such as mitochondria and chloroplasts. In plants, organellar introns often exhibit degenerate RNA structures, lacking essential elements for self-splicing and mobility. Consequently, their splicing relies heavily on host-encoded proteins. The plant organellar RNA recognition (PORR) domain, a recently identified RNA-binding motif, defines a small, plant-specific protein family. In this study, we characterized five novel mitochondria-targeted PORR genes. Functional analyses revealed that all five PORRs play a role in mitochondrial group II intron splicing. While one PORR protein was found necessary for the splicing of a single intron, the other four facilitate the splicing of multiple introns. Splicing defects in each porr mutant resulted in a significant reduction in complex I assembly and activity, along with an increase in the levels of other respiratory complexes. In vivo RNA binding studies revealed that the analyzed PORR proteins bind either to the 3′ end of trans-spliced intron 5′-halves or within intron domain I, indicating potential roles in trans-spliced intron rejoining or in the structural organization of domain I. Overall, these findings demonstrate that nuclear-encoded PORR proteins play crucial roles in the splicing of specific mitochondrial group II introns and are essential for mitochondrial biogenesis.

Group II introns in plant organelles have lost splicing autonomy and require the assistance of nuclear-encoded trans-factors whose roles remain to be elucidated. These factors can be mono- or poly-specific with respect to the number of introns whose splicing they facilitate. Poly-acting splicing factors are often essential and their genetic identification may benefit from the use of conditional mutations. Temperature-sensitive (TS) mutations in the ROOT PRIMORDIUM DEFECTIVE 1 (RPD1) gene were initially selected for their inhibitory effect on root formation in Arabidopsis. Further analysis revealed that RPD1 encodes a mitochondria-targeted RNA-binding protein family member, suggesting a role in mitochondrial gene expression and making its role in root formation enigmatic. We analysed the function of RPD1 and found that it is required for the removal of 9 mitochondrial group II introns and that the identified TS mutations affect the splicing function of RPD1. These results support that the inhibition of adventitious root formation at non-permissive temperature results from a reduction in RPD1 activity and thus mitochondrial activity. We further show that RPD1 physically associates in vivo with the introns whose splicing it facilitates. Preliminary mapping indicates that RPD1 may not bind to the same regions within all of its intron targets, suggesting potential variability in its influence on splicing activation.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that “mitochondrial translation factors” mTRAN1 and mTRAN2 are land plant–specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)–like RNA binding proteins of the mitoribosomal “small” subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5′ regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

Initiation and termination of plant mitochondrial transcription are poorly controlled steps. Precursor transcripts are thus often longer than necessary, and 3′-end processing as well as control of RNA stability are essential to produce mature mRNAs in plant mitochondria. Plant mitochondrial 3′ ends are determined by 3′-to-5′ exonucleolytic trimming until the progression of mitochondrial exonucleases along transcripts is stopped by stable RNA structures or RNA binding proteins. In this analysis, we investigated the function of the endonucleolytic mitochondrial stability factor 1 (EMS1) pentatricopeptide repeat (PPR) protein and showed that it is essential for the production and the stabilization of the mature form of the nad2 exons 1–2 precursor transcript, whose 3′ end corresponds to the 5′ half of the nad2 trans-intron 2. The accumulation of an extended rather than a truncated form of this transcript in ems1 mutant plants suggests that the role of EMS1 in 3′ end formation is not strictly limited to blocking the passage of 3′-5′ exonucleolytic activity, but that 3′ end formation of the nad2 exons 1–2 transcript involves an EMS1-dependent endonucleolytic cleavage. This study demonstrates that the formation of the 3′ end of mitochondrial transcripts may involve an interplay of endonucleolytic and exonucleolytic processing mediated by PPR proteins.

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmF_N2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmF_N2 and provides a stabilizing function for mitochondrial ribosomes.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the protein factors involved and the underlying molecular mechanisms are still not well understood. Here, we report on the function of the MITOCHONDRIAL STABILITY FACTOR 3 (MTSF3) protein, previously named EMBRYO DEFECTIVE 2794 (EMB2794), and show that it is essential for accumulation of the mitochondrial NADH dehydrogenase subunit 2 (nad2) transcript in Arabidopsis (Arabidopsis thaliana) but not for splicing of nad2 intron 2 as previously proposed. The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality, but viable mtsf3 mutant plants can be generated through partial complementation with the developmentally regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses revealed growth retardation in rescued mtsf3 plants owing to the specific destabilization of mature nad2 mRNA and a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3′ terminus of nad2. Destabilization of nad2 mRNA induces a substantial decrease in complex I assembly and activity and overexpression of the alternative respiratory pathway. Our results support a role for MTSF3 protein in protecting two nad2 transcripts from degradation by mitochondrial exoribonucleases by binding to their 3′ extremities.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the involved protein factors and the underlying molecular mechanisms are still not well understood. In this study, we report the function of the mitochondrial stability factor 3 (MTSF3) protein and we show that it is essential for accumulation of the mitochondrial nad2 transcript in Arabidopsis and not for the splicing of nad2 intron 2, as recently proposed (Marchetti et al., 2020). The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality but viable mtsf3 mutant plants could be generated by partial complementation with the developmentally-regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses reveal that mtsf3 rescued plants display growth retardation due to the specific destabilization of a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3’-terminus of nad2 . The destabilization of nad2 mRNA induces a significant decrease in complex I assembly and activity, and an overexpression of the alternative respiratory pathway. Our results support that the MTSF3 protein protects nad2 transcript from degradation by mitochondrial exoribonucleases by binding to its 3’ extremity.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players of plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo-development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1 and thus respiratory complex I biogenesis are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot-dicot divergence, ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.