New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.     

Multiple pathways influence mitochondrial inheritance in budding yeast

Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Delta and deletions of genes that affect mitochondrial function or inheritance, including mmr1Delta. Synthetic sickness of gem1Delta mmr1Delta double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution.     

The biology of yeast mitochondrial introns

Molecular Biology Reports -     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.     

Database for mobile group II introns

Group II introns are self-splicing RNAs and retroelements found in bacteria and lower eukaryotic organelles. During the past several years, they have been uncovered in surprising numbers in bacteria due to the genome sequencing projects; however, most of the newly sequenced introns are not correctly identified. We have initiated an ongoing web site database for mobile group II introns in order to provide correct information on the introns, particularly in bacteria. Information in the web site includes: (1) introductory information on group II introns; (2) detailed information on subfamilies of intron RNA structures and intron-encoded proteins; (3) a listing of identified introns with correct boundaries, RNA secondary structures and other detailed information; and (4) phylogenetic and evolutionary information. The comparative data should facilitate study of the function, spread and evolution of group II introns. The database can be accessed at http://www.fp.ucalgary.ca/group2introns/.     

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.     

DNA splicing of mitochondrial group I and II introns in Schizosaccharomyces pombe

Summary In this paper we report the precise excision of the group I intron aI2b from the cox1 gene and of the group II intron bI from the cob gene fo the Schizosaccharomyces pombe strain 50. We present evidence that DNA excision of both intron DNA sequences is under nuclear control. Attempts to remove the first cox1 intron (aI1) have failed so far, but a deletion of approximately 200 bp in the open intronic reading frame demonstrates that it is not essential for normal cellular functions.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome c oxidase - cob gene encoding apocytochrome b - rns and rnl genes encoding the small and large ribosomal RNA - atp6, atp8 and atp9 genes encoding subunits 6, 8, and 9 of the ATP synthase complex - urfa unassigned reading frame a - aI1, aI2a, aI2b, aI3 introns in the cox 1 gene of S. pombe - bI intron in the cob gene - del-aI2b and del-bI respiratory competent strains in which the respective introns have been deleted by DNA splicing     

Targeted inactivation of francisella tularensis genes by group II introns

Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.     

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by “subtractive genotyping”, to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.     

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.     

Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase.

bI4 maturase encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. It has been shown previously that a cytoplasmically translated hybrid protein composed of the pre-sequence of subunit 9 of Neurospora ATPase fused to a part of the bI4 maturase can be guided to mitochondria where it could compensate maturase deficiencies. This in vivo complementation of maturase mutants can be easily estimated by restoration of respiration. This work examines the efficiency of different bI4 maturase constructions to restore respiration in different yeast maturase-deficient strains. It is shown that the N-terminal end of the bI4 maturase plays a crucial role in the maturase activity. Moreover, the 12 N-terminal amino acids of the mitochondrial outer membrane protein constitute the most efficient mitochondrial targeting sequence in this system. Surprisingly enough, it was found that the cytoplasmically translated bI4 maturase containing the 254 C-terminal amino acid coded by the intron open reading frame can complement maturase mutations without any added mitochondrial-targeting sequence.     

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by subtractive genotyping, to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.