Fine mapping of the ribosomal RNA genes of HeLa cell mitochondrial DNA

A fine mapping study of the ribosomal RNA region of HeLa cell mitochondrial DNA has been carried out by using as an approach the protection by hybridized 12 S and 16 S rRNA of the complementary sequences in DNA against digestion with the single strand-specific Aspergillus nuclease S₁ or Escherichia coli exonuclease VII. No inserts have been detected in the main body of the 12 S and 16 S rRNA cistrons, in contrast to the situation described in the large mitochondrial ribosomal RNA gene of some strains of yeast and of Neurospora crassa. Furthermore, it has been possible to assign more precisely than previously the positions of the 5′ and 3′-ends of the 12 S rRNA and 16 S rRNA genes in the HpaII restriction map of HeLa cell mitochondrial DNA.     

Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei

Mitochondrial RNA turnover in yeast involves the degradosome, composed of DSS-1 exoribonuclease and SUV3 RNA helicase. Here, we describe a degradosome-like complex, containing SUV3 and DSS-1 homologues, in the early branching protozoan, Trypanosoma brucei. TbSUV3 is mitochondrially localized and co-sediments with TbDSS-1 on glycerol gradients. Co-immunoprecipitation demonstrates that TbSUV3 and TbDSS-1 associate in a stable complex, which differs from the yeast degradosome in that it is not stably associated with mitochondrial ribosomes. This is the first report of a mitochondrial degradosome-like complex outside of yeast. Our data indicate an early evolutionary origin for the mitochondrial SUV3/DSS-1 containing complex.

Structured summary

MINT-7187980: SUV3 (genbank_protein_gi:XP_844349) and DSS1 (uniprotkb:Q38EM3) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7188074: SUV3 (genbank_protein_gi:XP_844349) physically interacts (MI:0914) with DSS1 (uniprotkb:Q38EM3) by anti tag co-immunoprecipitation (MI:0007)     

The yeast mitochondrial degradosome. Its composition,interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism

The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism. Previous purifications suggested that it was composed of three subunits. Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes. We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3'- and 5'-ends. The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing. We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.     

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU intron in vivo

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiaeCBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiaeCBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron.     

Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3'' end of the gene.

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.     

An intron in a ribosomal protein gene from Tetrahymena

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.     

Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions

A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome. The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method. The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5′ and 3′ ends of the maize 26 S rDNA have been located by comparison with the E. coli sequence. The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA. In all cases, there are numerous regions of 70% or higher homology.     

Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm⁵s²U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P^S) or 15S rRNA C1409G allele (P^R) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA^Lys, tRNA^Glu, tRNA^Gln, tRNA^Leu, tRNA^Gly, tRNA^Arg and tRNA^Phe were decreased significantly while those of tRNA^Met and tRNA^His were not affected in the mto1 strains carrying the P^S allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA^Lys, tRNA^Met and tRNA^Phe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA^Lys, tRNA^Leu, tRNA^Arg but almost no effect in those of tRNA^His. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.     

The role of a conserved dodecamer sequence in yeast mitochondrial gene expression

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.     

Nature of an inserted sequence in the mitochondrial gene coding for the 15S ribosomal RNA of yeast

The small ribosomal RNA, or 15S RNA, or yeast mitochondria is coded by a mitochondrial gene. In the central part of the gene, there is a guanine-cytosine (GC) rich sequence of 40 base-pairs, flanked by adenine-thymine sequences. The GC-rich sequence is (5') TAGTTCCGGGGCCCGGCCACGGAGCCGAACCCGAAAGGAG (3'). We have found that this sequence is absent in the 15S rRNA gene of some strains of yeast. When present, it is transcribed into the mature 15S rRNA to produce a longer variant of the RNA. Sequences identical or closely related to this GC-rich sequence are present in many regions of the mitochondrial genome of Saccharomyces cerevisiae. The 5' and 3' terminal structures of all these sequences are highly constant.