Temperature-sensitive splicing defects in Arabidopsis mitochondria caused by mutations in the ROOT PRIMORDIUM DEFECTIVE 1 gene.

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.

Interplay of endonucleolytic and exonucleolytic processing in the 3'-end formation of a mitochondrial nad2 RNA precursor in Arabidopsis.

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.

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA.

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138 We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

Pentatricopeptide repeat protein MITOCHONDRIAL STABILITY FACTOR 3 ensures mitochondrial RNA stability and embryogenesis.

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.

Rerouting of ribosomal proteins into splicing in plant organelles.

Production and expression of RNA requires the action of multiple RNA-binding proteins (RBPs). New RBPs are most often created by novel combinations of dedicated RNA-binding modules. However, recruiting existing genes to create new RBPs is also an important evolutionary strategy. In this report, we analyzed the eight-member uL18 ribosomal protein family in Arabidopsis uL18 proteins share a short structurally conserved domain that binds the 5S ribosomal RNA (rRNA) and allows its incorporation into ribosomes. Our results indicate that Arabidopsis uL18-Like proteins are targeted to either mitochondria or chloroplasts. While two members of the family are found in organelle ribosomes, we show here that two uL18-type proteins function as factors necessary for the splicing of certain mitochondrial and plastid group II introns. These two proteins do not cosediment with mitochondrial or plastid ribosomes but instead associate with the introns whose splicing they promote. Our study thus reveals that the RNA-binding capacity of uL18 ribosomal proteins has been repurposed to create factors that facilitate the splicing of organellar introns.

The pentatricopeptide repeat protein MTSF2 stabilizes a nad1 precursor transcript and defines the 3΄ end of its 5΄-half intron.

RNA expression in plant mitochondria implies a large number of post-transcriptional events in which transcript processing and stabilization are essential. In this study, we analyzed the function of the Arabidopsis mitochondrial stability factor 2 gene (MTSF2) and show that the encoded pentatricopeptide repeat protein is essential for the accumulation of stable nad1 mRNA. The production of mature nad1 requires the assembly of three independent RNA precursors via two trans-splicing reactions. Genetic analyses revealed that the lack of nad1 in mtsf2 mutants results from the specific destabilization of the nad1 exons 2-3 precursor transcript. We further demonstrated that MTSF2 binds to its 3΄ extremity with high affinity, suggesting a protective action by blocking exoribonuclease progression. By defining the 3΄ end of nad1 exons 2-3 precursor, MTSF2 concomitantly determines the 3΄ extremity of the first half of the trans-intron found at the end of the transcript. Therefore, binding of the MTSF2 protein to nad1 exons 2-3 precursor evolved both to stabilize the transcript and to define a 3΄ extremity compatible with the trans-splicing reaction needed to reconstitute mature nad1. We thus reveal that the range of transcripts stabilized by association with protective protein on their 3΄ end concerns also mitochondrial precursor transcripts.

Three new pentatricopeptide repeat proteins facilitate the splicing of mitochondrial transcripts and complex I biogenesis in Arabidopsis.

Group II introns are common features of most angiosperm mitochondrial genomes. Intron splicing is thus essential for the expression of mitochondrial genes and is facilitated by numerous nuclear-encoded proteins. However, the molecular mechanism and the protein cofactors involved in this complex process have not been fully elucidated. In this study, we characterized three new pentatricopeptide repeat (PPR) genes, called MISF26, MISF68, and MISF74, of Arabidopsis and showed they all function in group II intron splicing and plant development. The three PPR genes encode P-type PPR proteins that localize in the mitochondrion. Transcript analysis revealed that the splicing of a single intron is altered in misf26 mutants, while several mitochondrial intron splicing defects were detected in misf68 and misf74 mutants. To our knowledge, MISF68 and MISF74 are the first two PPR proteins implicated in the splicing of more than one intron in plant mitochondria, suggesting that they may facilitate splicing differently from other previously identified PPR splicing factors. The splicing defects in the misf mutants induce a significant decrease in complex I assembly and activity, and an overexpression of mRNAs of the alternative respiratory pathway. These results therefore reveal that nuclear encoded proteins MISF26, MISF68, and MISF74 are involved in splicing of a cohort of mitochondrial group II introns and thereby required for complex I biogenesis.

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

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.

[Microbial Diversity of Filamentous Sludge Bulking at Low Temperature].

To investigate the characteristics of microbial diversity during filamentous bulking at low temperature, the induction of sludge bulking was successfully carried out using a low-temperature sequencing batch reactor(SBR). With the help of Illumina MiSeq high-throughput sequencing technology, the overall changes in the microbial community structure of activated sludge, the characteristics of each specific microbial community, and the specific genera were all investigated under different sludge sedimentation performances. The results showed that filamentous bulking can be successfully induced after the system operating temperature drops to (14±1)℃, and the COD and TN removal rates can still be maintained at approximately 90% and 86%, respectively, with the sludge volume index deteriorating to 663.99 mL·g-1. The occurrence of sludge bulking at low temperature will not only reduce the overall diversity and uniformity of microorganisms in the system and increase the abundance of filamentous bacteria from 0.49% to 26.04% but also cause the abundance of denitrifying bacteria to reduce from 21.04% to 13.99% and that of dephosphorization bacteria to reduce from 4.25% to 1.93%. Of the five filamentous genera founded, the abundances of three filamentous bacteria represented by Thiothrix increased, whereas only that of the Haliscomenobacter decreased. Of the 19 denitrifier genera founded, the abundances of five species represented by Nitrosomonas increased, whereas those of seven species represented by Nitrospira decreased. Moreover, the abundances of Pseudomonas and Tetrasphaera increased out of the eight phosphorus-removing bacteria genera, whereas the abundances of the five bacteria genera represented by Candidatus_Competibacter decreased. Although sludge bulking has a significant impact on the structure of the microbial community, the 477 operational taxonomic units and 227 bacterial species that are always present in the different sludge samples indicate that the main microorganisms in the reactor are still relatively stable during the bulking process.