Shifts from cis-to trans-splicing of five mitochondrial introns in Tolypanthus maclurei

Shifts from cis-to trans-splicing of mitochondrial introns tend to correlate with relative genome rearrangement rates during vascular plant evolution, as is particularly apparent in some lineages of gymnosperms. However, although many angiosperms have also relatively high mitogenomic rearrangement rates, very few cis-to trans-splicing shifts except for five trans-spliced introns shared in seed plants have been reported. In this study, we sequenced and characterized the mitogenome of Tolypanthus maclurei, a hemiparasitic plant from the family Loranthaceae (Santalales). The mitogenome was assembled into a circular chromosome of 256,961 bp long, relatively small compared with its relatives from Santalales. It possessed a gene content of typical angiosperm mitogenomes, including 33 protein-coding genes, three rRNA genes and ten tRNA genes. Plastid-derived DNA fragments took up 9.1% of the mitogenome. The mitogenome contained one group I intron (cox1i729) and 23 group II introns. We found shifts from cis-to trans-splicing of five additional introns in its mitogenome, of which two are specific in T. maclurei. Moreover, atp1 is a chimeric gene and phylogenetic analysis indicated that a 356 bp region near the 3′ end of atp1 of T. maclurei was acquired from Lamiales via horizontal gene transfer. Our results suggest that shifts to trans-splicing of mitochondrial introns may not be uncommon among angiosperms.

Recent and Ongoing Horizontal Transfer of Mitochondrial Introns Between Two Fungal Tree Pathogens

Two recently introduced fungal plant pathogens (Ceratocystis lukuohia and Ceratocystis huliohia) are responsible for Rapid ‘ōhi‘a Death (ROD) in Hawai‘i. Despite being sexually incompatible, the two pathogens often co-occur in diseased ‘ōhi‘a sapwood, where genetic interaction is possible. We sequenced and annotated 33 mitochondrial genomes of the two pathogens and related species, and investigated 35 total Ceratocystis mitogenomes. Ten mtDNA regions [one group I intron, seven group II introns, and two autonomous homing endonuclease (HE) genes] were heterogeneously present in C. lukuohia mitogenomes, which were otherwise identical. Molecular surveys with specific primers showed that the 10 regions had uneven geographic distribution amongst populations of C. lukuohia. Conversely, identical orthologs of each region were present in every studied isolate of C. huliohia regardless of geographical origin. Close relatives of C. lukuohia lacked or, rarely, had few and dissimilar orthologs of the 10 regions, whereas most relatives of C. huliohia had identical or nearly identical orthologs. Each region included or worked in tandem with HE genes or reverse transcriptase/maturases that could facilitate interspecific horizontal transfers from intron-minus to intron-plus alleles. These results suggest that the 10 regions originated in C. huliohia and are actively moving to populations of C. lukuohia, perhaps through transient cytoplasmic contact of hyphal tips (anastomosis) in the wound surface of ‘ōhi‘a trees. Such contact would allow for the transfer of mitochondria followed by mitochondrial fusion or cytoplasmic exchange of intron intermediaries, which suggests that further genomic interaction may also exist between the two pathogens.

Empty Pericarp24 and Empty Pericarp25 Are Required for the Splicing of Mitochondrial Introns, Complex I Assembly, and Seed Development in Maize

RNA splicing is an essential post-transcriptional regulation in plant mitochondria and chloroplasts. As the mechanism of RNA splicing remains obscure, identification and functional elucidation of new splicing factors are necessary. Through a characterization of two maize mutants, we cloned Empty pericarp 24 (Emp24) and Empty pericarp 25 (Emp25). Both Emp24 and Emp25 encode mitochondrion-targeted P-type PPR proteins. EMP24 is required for the splicing of nad4 introns 1 and 3, which was reported (Ren Z. et al., 2019), and EMP25 functions in the splicing of nad5 introns 1, 2, and 3. Absence of either Nad4 or Nad5 proteins blocks the assembly of mitochondrial complex I, resulting in the formation of a sub-sized complex I of similar size in both mutants. Mass spectrometry identification revealed that the subcomplexes in both mutants lack an identical set of proteins of complex I. These results indicate that EMP24 and EMP25 function in the splicing of nad4 and nad5 introns, respectively, and are essential to maize kernel development. The identification of the subcomplexes provides genetic and molecular insights into the modular complex I assembly pathway in maize.

nMAT3 is an essential maturase splicing factor required for holo-complex I biogenesis and embryo-development in Arabidopsis thaliana plants

SummaryGroup II introns are large catalytic RNAs that are particularly prevalent in the organelles of terrestrial plants. In angiosperm mitochondria, group II introns reside in the coding-regions of many critical genes, and their excision is essential for respiratory-mediated functions. Canonical group II introns are self-splicing and mobile genetic elements, consisting of the catalytic intron-RNA and its cognate intron-encoded endonuclease factor (i.e. maturase, Pfam-PF01348). Plant organellar introns are extremely degenerate, and lack many regions that are critical for splicing, including their related maturase-ORFs. The high degeneracy of plant mitochondrial introns was accompanied during evolution by the acquisition of ‘host-acting’ protein cofactors. These include several nuclear encoded maturases (nMATs) and various other splicing-cofactors that belong to a diverse set of RNA-binding families, e.g. RNA helicases (Pfam-PF00910), Mitochondrial Transcription Termination Factors (mTERF, Pfam-PF02536), Plant Organelle RNA Recognition (PORR, Pfam-PF11955), and Pentatricopeptide repeat (PPR, Pfam-PF13812) proteins. Previously, we established the roles of MatR and three nuclear-maturases, nMAT1, nMAT2, and nMAT4, in the splicing of different subsets of mitochondrial introns in Arabidopsis. The function of nMAT3 (AT5G04050) was found to be essential during early embryogenesis. Using a modified embryo-rescue method, we show that nMAT3-knockout plants are strongly affected in the splicing of nad1 introns i1, i3 and i4 in Arabidopsis mitochondria. The embryo-defect phenotype is tightly associated with complex I biogenesis defects. Functional complementation of nMAT3 restored the splicing defects and altered embryogenesis phenotypes associated with the nmat3 mutant-line.

Schizosaccharomyces pombe Ppr10 is required for mitochondrial translation

ABSTRACT The mitochondrial genome encodes key components of the oxidative phosphorylation (OXPHOS) system, whose expression is essential for mitochondrial functions. We have previously shown that deletion of the Schizosaccharomyces pombe ppr10 encoding a pentatricopeptide repeat protein severely reduces the mature levels of intron-containing mitochondrial transcripts cox1 and cob1, and severely impairs mitochondrial translation. In this study, we examined the possibility that the reduced levels of Cox1 and Cob1 proteins in cells were due to lowered levels of cox1 and cob1 mRNAs. We found that deletion of ppr10 did not affect the levels of mature cox1 and cob1 mRNAs in a mitochondrial intronless background. However, synthesis of Cox1 and Cob1 proteins were still severely affected by deletion of ppr10 in a mitochondrial intronless background. Consistent with this, we found that deletion of mitochondrial introns could not rescue the respiratory growth defect of Δppr10 cells. Our results reveal that Ppr10 is not required for the stability of cox1 and cob1 mRNAs, and provide further support for the idea that Ppr10 plays a critical role in mitochondrial translation.

Extensive Shifts from Cis- to Trans-splicing of Gymnosperm Mitochondrial Introns

Hundreds of plant mitogenomes have been sequenced from angiosperms, but relatively few mitogenomes are available from its sister lineage, gymnosperms. To examine mitogenomic diversity among extant gymnosperms, we generated draft mitogenomes from 11 diverse species and compared them with four previously published mitogenomes. Examined mitogenomes from Pinaceae and cycads retained all 41 protein genes and 26 introns present in the common ancestor of seed plants, whereas gnetophyte and cupressophyte mitogenomes experienced extensive gene and intron loss. In Pinaceae and cupressophyte mitogenomes, an unprecedented number of exons are distantly dispersed, requiring trans-splicing of 50–70% of mitochondrial introns to generate mature transcripts. RNAseq data confirm trans-splicing of these dispersed exons in Pinus. The prevalence of trans-splicing in vascular plant lineages with recombinogenic mitogenomes suggests that genomic rearrangement is the primary cause of shifts from cis- to trans-splicing in plant mitochondria.

Proposal of a new nomenclature for introns in protein-coding genes in fungal mitogenomes

Fungal mitochondrial genes are often invaded by group I or II introns, which represent an ideal marker for understanding fungal evolution. A standard nomenclature of mitochondrial introns is needed to avoid confusion when comparing different fungal mitogenomes. Currently, there has been a standard nomenclature for introns present in rRNA genes, but there is a lack of a standard nomenclature for introns present in protein-coding genes. In this study, we propose a new nomenclature system for introns in fungal mitochondrial protein-coding genes based on (1) three-letter abbreviation of host scientific name, (2) host gene name, (3), one capital letter P (for group I introns), S (for group II introns), or U (for introns with unknown types), and (4) intron insertion site in the host gene according to the cyclosporin-producing fungus Tolypocladium inflatum. The suggested nomenclature was proved feasible by naming introns present in mitogenomes of 16 fungi of different phyla, including both basal and higher fungal lineages although minor adjustment of the nomenclature is needed to fit certain special conditions. The nomenclature also had the potential to name plant/protist/animal mitochondrial introns. We hope future studies follow the proposed nomenclature to ensure direct comparison across different studies.

CFM9, a Mitochondrial CRM Protein, Is Crucial for Mitochondrial Intron Splicing, Mitochondria Function and Arabidopsis Growth and Stress Responses

Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.

Intraspecific Diversity of Fission Yeast Mitochondrial Genomes

The fission yeast Schizosaccharomyces pombe is an important model organism, but its natural diversity and evolutionary history remain under-studied. In particular, the population genomics of the S. pombe mitochondrial genome (mitogenome) has not been thoroughly investigated. Here, we assembled the complete circular-mapping mitogenomes of 192 S. pombe isolates de novo, and found that these mitogenomes belong to 69 nonidentical sequence types ranging from 17,618 to 26,910 bp in length. Using the assembled mitogenomes, we identified 20 errors in the reference mitogenome and discovered two previously unknown mitochondrial introns. Analyzing sequence diversity of these 69 types of mitogenomes revealed two highly distinct clades, with only three mitogenomes exhibiting signs of inter-clade recombination. This diversity pattern suggests that currently available S. pombe isolates descend from two long-separated ancestral lineages. This conclusion is corroborated by the diversity pattern of the recombination-repressed K-region located between donor mating-type loci mat2 and mat3 in the nuclear genome. We estimated that the two ancestral S. pombe lineages diverged about 31 million generations ago. These findings shed new light on the evolution of S. pombe and the data sets generated in this study will facilitate future research on genome evolution.

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

Two maize nucleus-encoded splicing factors, PPR-SMR1 and Zm-mCSF1, are required for the splicing of most mitochondrial group II introns and subsequent complex I biogenesis, and therefore play important roles in seed development.