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.     

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.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

Evolution of Plant Mitochondrial Intron-Encoded Maturases: Frequent Lineage-Specific Loss and Recurrent Intracellular Transfer to the Nucleus

Among land plants, mitochondrial and plastid group II introns occasionally encode proteins called maturases that are important for splicing. Angiosperm nuclear genomes also encode maturases that are targeted to the organelles, but it is not known whether nucleus-encoded maturases exist in other land plant lineages. To examine the evolutionary diversity and history of this essential gene family, we searched for maturase homologs in recently sequenced nuclear and mitochondrial genomes from diverse land plants. We found that maturase content in mitochondrial genomes is highly lineage specific, such that orthologous maturases are rarely shared among major land plant groups. The presence of numerous mitochondrial pseudogenes in the mitochondrial genomes of several species implies that the sporadic maturase distribution is due to frequent inactivation and eventual loss over time. We also identified multiple maturase paralogs in the nuclear genomes of the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens, and the representative angiosperm Vitis vinifera. Phylogenetic analyses of organelle- and nucleus-encoded maturases revealed that the nuclear maturase genes in angiosperms, lycophytes, and mosses arose by multiple shared and independent transfers of mitochondrial paralogs to the nuclear genome during land plant evolution. These findings indicate that plant mitochondrial maturases have experienced a surprisingly dynamic history due to a complex interaction of multiple evolutionary forces that affect the rates of maturase gain, retention, and loss.     

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize(Zea mays) DEAD-box RNA helicase48(Zm RH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis,and seed development. Loss of Z...     

The Mitochondrial Genome of Chlorogonium elongatum Inferred from the Complete Sequence

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA. Received: 3 November 1997 / Accepted: 12 January 1998     

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.     

The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns

Summary The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473–483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%–50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2 ^– mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.     

Trans-splicing group II introns in plant mitochondria: the complete set of cis-arranged homologs in ferns, fern allies, and a hornwort.

The fragmentation of group II introns without concomitant loss of splicing competence is illustrated by extraordinary gene arrangements in plant mitochondrial genomes. The mitochondrial genes nad1, nad2, and nad5, all encoding subunits of the NADH dehydrogenase, require trans-splicing for functional assembly of their mRNAs in flowering plants. Tracing the origins of trans-splicing group II introns shows that they have evolved from formerly cis-arranged homologs whose descendants can still be identified in lineages of early branching land plants. In this contribution we present the full set of ancestor introns for all five conserved mitochondrial trans-splicing positions. These introns are strikingly small in the quillwort Isoetes lacustris, the continuous nad2 gene intron in this species representing the smallest (389 nt) land plant group II intron yet identified. cDNA analysis shows correct splicing of the introns in vivo and also identifies frequent RNA editing events in the flanking nad gene exons. Other representatives of the ancestral cis-arranged introns are identified in the fern Osmunda regalis, the horsetail Equisetum telmateia, and the hornwort Anthoceros crispulus. Only the now identified intron in Osmunda carries significant traces of a former maturase reading frame. The identification of a continuous homolog in Anthoceros demonstrates that intron invasion into the affected genes in some cases predated the split of vascular and nonvascular plants more than 400 million years ago. As an alternative to disruption after size increase, the respective introns can get secondarily lost in certain lineages.     

Evolution from DNA to RNA recognition by the bI3 LAGLIDADG maturase

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.     

A PORR domain protein required for rpl2 and ccmF(C) intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria

Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus‐encoded splicing factors have been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified: rpl2 and ccmF_C. WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in wtf9 mutants as a means to functionally characterize the mitochondrial rpl2 and ccmF_C genes. Loss of ccmF_C expression correlates with the loss of cytochromes c and c₁, confirming a role for ccmF_C in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial rpl2 gene, and imply that the Rpl2 fragment encoded by rpl2 exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein in trans.