nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.     

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis

Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membranebound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants’ seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants.     

Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome

Complex I deficiency, the most common cause of mitochondrial disorders, accounts for a variety of clinical symptoms and its genetic heterogeneity makes identification of the disease genes particularly tedious. Indeed, most of the 43 complex I subunits are encoded by nuclear genes, only seven of them being mitochondrially encoded. In order to offer urgent prenatal diagnosis, we have studied an inbred/multiplex family with complex I deficiency by using microsatellite DNA markers flanking the putative disease loci. Microsatellite DNA markers have allowed us to exclude the NDUFS7, NDUFS8, NDUFV1 and NDUFS1 genes and to find homozygosity at the NDUFS4 locus. Direct sequencing has led to identification of a homozygous splice acceptor site mutation in intron 1 of the NDUFS4 gene (IVS1nt -1, G-->A); this was not found in chorion villi of the ongoing pregnancy. We suggest that genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families helps to identify the disease-causing mutation. More generally, we suggest giving consideration to a more systematic microsatellite analysis of putative disease loci for identification of disease genes in inbred/multiplex families affected with genetically heterogeneous conditions.     

Trans splicing in Oenothera mitochondria: nad1 mRNAs are edited in exon and trans-splicing group II intron sequences.

The complete NADH dehydrogenase subunit 1 (nad1) ORF in Oenothera mitochondria is encoded by five exons. These exons are located in three distant locations of the mitochondrial genome. One genomic region encodes exon a, the second encodes exons b and c, and the third specifies exons d and e. Cis-splicing group II introns separate exons b and c and d and e, while trans-splicing reactions are required to link exons a and b and c and d. The two parts of the group II intron sequences involved in these trans-splicing events can be aligned in domain IV. Exon sequences and the maturase-related ORF in intron d/e are edited by numerous C to U alterations in the mRNA. Two RNA editing events in the trans-splicing intron a/b improve conservation of the secondary structure in the stem of domain VI. RNA editing in intron sequences may thus be required for the trans-splicing reaction.     

nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation,is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self‐splicing ribozyme and its own intron‐encoded maturase protein. A hallmark of maturases is that they are intron‐specific, acting as cofactors that bind their intron‐containing pre‐RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron‐encoded maturase open reading frames suggest that their splicing in vivo is assisted by ‘trans’‐acting protein factors. Interestingly, angiosperms harbor several nuclear‐encoded maturase‐related (nMat) genes that contain N‐terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.     

A molecular link between mitochondrial preprotein transporters and respiratory chain complexes

The TIM17:23 complex on the mitochondrial inner membrane is responsible for import of the majority of mitochondrial proteins in plants. In Arabidopsis, Tim17 and Tim23 belong to a large gene family consisting of 16 members termed the Preprotein and Amino acid transporters (PRAT). Recently, two members of this protein family, Tim23-2 and the Complex I subunit B14.7, have been shown to assemble into both Complex I of the respiratory chain and the TIM17:23 complex (Wang et al., 2012), adding to other examples of links between respiratory and protein import complexes. These associations provide a mechanism to coordinate mitochondrial activity and biogenesis.