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.     

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.     

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.     

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...     

Variation in sequence and RNA editing within core domains of mitochondrial group II introns among plants

The 3' regions of several group II introns within the mitochondrial genes nad1 and nad7 show unexpected sequence divergence among flowering plants, and the core domains 5 and 6 are predicted to have weaker helical structure than those in self-splicing group II introns. To assess whether RNA editing improves helical stability by the conversion of A-C mispairs to A-U pairs, we sequenced RT-PCR amplification products derived from excised intron RNAs or partially spliced precursors. Only in some cases was editing observed to strengthen the predicted helices. Moreover, the editing status within nad1 intron 1 and nad7 intron 4 was seen to differ among plant species, so that homologous intron sequences shared lower similarity at the RNA level than at the DNA level. Plant-specific variation was also seen in the length of the linker joining domains 5 and 6 of nad7 intron 3; it ranged from 4 nt in wheat to 11 nt in soybean, in contrast to the 2-4 nt length typical of classical group II introns. However, this intron is excised as a lariat structure with a domain 6 branchpoint adenosine. Our observations suggest that the core structures and sequences of these plant mitochondrial introns are subject to less stringent evolutionary constraints than conventional group II introns.     

A latent intron-encoded maturase is also an endonuclease needed for intron mobility

Some yeast mitochondrial introns encode proteins that promote either splicing (maturases) or intron propagation via gene conversion (the fit1 endonuclease). We surveyed introns in the coxl gene for their ability to engage in gene conversion and found that the group I intron, al4 alpha, was efficiently transmitted to genes lacking it. An endonucleolytic cleavage is detectable in recipient DNA molecules near the site of intron insertion in vivo and in vitro. Conversion is dependent on an intact al4 alpha open reading frame. This intron product is a latent maturase, but these data show that it is also a potent endonuclease involved in recombination. Dual function proteins that cleave DNA and facilitate RNA splicing may have played a pivotal role in the propagation and tolerance of introns.     

The complete mitochondrial DNA sequence of Mesostigma viride identifies this green alga as the earliest green plant divergence and predicts a highly compact mitochondrial genome in the ancestor of all green plants.

To gain insights into the nature of the mitochondrial genome in the common ancestor of all green plants, we have completely sequenced the mitochondrial DNA (mtDNA) of Mesostigma viride. This green alga belongs to a morphologically heterogeneous class (Prasinophyceae) that includes descendants of the earliest diverging green plants. Recent phylogenetic analyses of ribosomal RNAs (rRNAs) and concatenated proteins encoded by the chloroplast genome identified Mesostigma as a basal branch relative to the Streptophyta and the Chlorophyta, the two phyla that were previously thought to contain all extant green plants. The circular mitochondrial genome of Mesostigma resembles the mtDNAs of green algae occupying a basal position within the Chlorophyta in displaying a small size (42,424 bp) and a high gene density (86.6% coding sequences). It contains 65 genes that are conserved in other mtDNAs. Although none of these genes represents a novel coding sequence among green plant mtDNAs, four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs, and two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs examined so far (land-plant mtDNAs). Phylogenetic analyses of 19 concatenated mtDNA-encoded proteins favor the hypothesis that Mesostigma represents the earliest branch of green plant evolution. Four group I introns (two in rnl and two in cox1) and three group II introns (two in nad3 and one in cox2), two of which are trans-spliced at the RNA level, reside in Mesostigma mtDNA. The insertion sites of the three group II introns are unique to this mtDNA, suggesting that trans-splicing arose independently in the Mesostigma lineage and in the Streptophyta. The few structural features that can be regarded as ancestral in Mesostigma mtDNA predict that the common ancestor of all green plants had a compact mtDNA containing a minimum of 75 genes and perhaps two group I introns. Considering that the mitochondrial genome is much larger in size in land plants than in Mesostigma, we infer that mtDNA size began to increase dramatically in the Streptophyta either during the evolution of charophyte green algae or during the transition from charophytes to land plants.     

Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.     

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.