Neurospora crassa mitochondrial transfer RNAs.

Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tRNA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found psi, T, hU, t6A, m1G, M2G, m22G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electropherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

Multiple specificity recognition motifs enhance plant mitochondrial RNA editing in vitro

Analysis of RNA editing in plant mitochondria has at least in vitro been hampered by very low activity. Consequently, none of the trans-acting factors involved has yet been identified. We here report that in vitro RNA editing increases dramatically when additional cognate recognition motifs are introduced into the template RNA molecule. Substrate RNAs with tandemly repeated recognition elements enhance in vitro RNA editing from 2-3% to 50-80%. The stimulation is not influenced by the editing status of a respective RNA editing site, suggesting that specific recognition of a site can be independent of the edited nucleotide itself. In vivo, attachment of the editing complex may thus be analogously initiated at sequence similarities in the vicinity of bona fide editing sites. This cis-acting enhancement decreases with increasing distance between the duplicated specificity signals; a cooperative effect is detectable up to approximately 200 nucleotides. Such repeated template constructs promise to be powerful tools for the RNA affinity identification of the as yet unknown trans-factors of plant mitochondrial RNA editing.     

Computational analysis of RNA editing sites in plant mitochondrial genomes reveals similar information content and a sporadic distribution of editing sites

A computational analysis of RNA editing sites was performedon protein-coding sequences of plant mitochondrial genomes fromArabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryzasativa. The distribution of nucleotides around edited and uneditedcytidines was compared in 41 nucleotide segments and included1481 edited cytidines and 21,390 unedited cytidines in the 4genomes. The distribution of nucleotides was examined in 1,2, and 3 nucleotide windows by comparison of nucleotide frequencyratios and relative entropy. The relative entropy analyses indicatethat information is encoded in the nucleotide sequences in the5 prime flank (–18 to –14, –13 to –10,–6 to –4, –2/–1) and the immediate 3prime flanking nucleotide (+1), and these regions may be importantin editing site recognition. The relative entropy was largewhen 2 or 3 nucleotide windows were analyzed, suggesting thatseveral contiguous nucleotides may be involved in editing siterecognition. RNA editing sites were frequently preceded by 2pyrimidines or AU and followed by a guanidine (HYCG) in themonocot and dicot mitochondrial genomes, and rarely precededby 2 purines. Analysis of chloroplast editing sites from a dicot,Nicotiana tabacum, and a monocot, Zea mays, revealed a similardistribution of nucleotides around editing sites (HYCA). Thesimilarity of this motif around editing sites in monocots anddicots in both mitochondria and chloroplasts suggests that amechanistic basis for this motif exists that is common in thesedifferent organelle and phylogenetic systems. The preferredsequence distribution around RNA editing sites may have an importantimpact on the acquisition of editing sites in evolution becausethe immediate sequence context of a cytidine residue may rendera cytidine editable or uneditable, and consequently determinewhether a T to C mutation at a specific position may be correctedby RNA editing. The distribution of editing sites in many protein-codingsequences is shown to be non-random with editing sites clusteredin groups separated by regions with no editing sites. The sporadicdistribution of editing sites could result from a mechanismof editing site loss by gene conversion utilizing edited sequenceinformation, possibly through an edited cDNA intermediate.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

Analysis of the regions coding for transfer RNAs in Kluyveromyces lactis mitochondrial DNA.

The major regions coding for the transfer RNA genes in the mitochondrial DNA of K. lactis were studied. Twenty one, out of a supposed twenty four tRNA genes were identified and localized with respect to other mitochondrial genes. Most of the tRNA genes were found in a cluster downstream of the large ribosomal RNA gene. The order of a few groups of genes is conserved with respect to S. cerevisiae and T. glabrata. The highly diverged intergenic sequences contained a large number of guanine-cytosine clusters which frequently formed long palindromic sequences.     

Mammalian mitochondrial transfer RNAs: chromatographic properties, size and origin.

Incubation of isolated rat liver mitochondria with radioactive amino acids resulted in the charging of tRNAs for arginine, asparagine, leucine, lysine, methionine, proline and valine. The aminoacyl-tRNAs were shown to be distinct from their cytosolic counterparts by chromatography on RPC-5. By electrophoresis on urea polyacrylamide slab gels it was found that all these mitochondrial aminoacyl-tRNAs were about 70-76 nucleotides long. The unique mitochondrial asparaginyl- and prolyl-tRNAs, not previously identified in mammalian cells, were shown to hybridize to mtDNA. Mitochondrial leucyl-tRNA separated into 3 peaks on RPC-5 and the first species was shown to be different than a combination of the other two by molecular size and partial RNase T1 digestion patterns. Each was coded by a separate gene on mtDNA as shown by partial additivity of hybridization. Separate genes for mitochondrial tRNAMetm and tRNAMetf, separated by RPC-5 chromatography, were also demonstrated. These results bring to 21 the number of individual tRNAs coded by mammalian mtDNA.     

Mutation accumulation in transfer RNAs: molecular evidence for Muller's ratchet in mitochondrial genomes

The accumulation of deleterious mutations is thought to be a major factor preventing the long-term persistence of obligately asexual lineages relative to their sexual ancestors. This phenomenon is also of potential relevance to sexual species that harbor asexually propagating organelle genomes. A comparative study of the transfer RNA genes in animal mitochondrial and nuclear genomes demonstrates that the former accumulate nucleotide substitutions much more rapidly than do the latter, and several lines of evidence are consistent with the idea that the excess substitutions are mildly deleterious. First, the average binding stability between complementary strands in the stems of mitochondrial tRNAs is less than half that in nuclear tRNAs. Second, most loop sizes in the mitochondrial tRNAs have experienced a net reduction in size over evolutionary time, and they are nearly 50 times more variable in the mitochondrial than in the nuclear genome. Third, although nearly 20% of the nucleotides in nuclear tRNA genes (particularly those involved in tertiary interactions) are invariant across all animal taxa and all tRNA species, there are no invariant sites in the mitochondrial tRNAs. These observations, as well as results from recent laboratory experiments, are consistent with the hypothesis that nonrecombining organelle genomes are subject to gradual loss of fitness due to the cumulative chance fixation of mildly deleterious mutations.      

RNA editing in plant mitochondria

RNA editing in plant mitochondria alters nearly all mRNAs by C to U and U to C transitions. In some species more than 400 edited sites have been identified with significant effects on the encoded proteins. RNA editing occurs in higher and lower plants and presumably has evolved before the differentiation of land plants. Current research focuses on the elucidation of the biochemistry and the specificity determinants of RNA editing in plant mitochrondria.     

Role of plant lipid transfer proteins in plant cell physiology-a concise review

Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.