ABSTRACT
RNA editing is unique among post-transcriptional processes in plastids, as it exhibits extraordinary phylogenetic dynamics leading to species-specific editing site patterns. The evolutionary loss of a site is considered to entail the loss of the corresponding nuclear-encoded site-specific factor, which prevents the editing of foreign, i.e. heterologous, sites. We investigated the editing of short 'spliced' and 'unspliced' ndhA gene fragments from spinach in Nicotiana tabacum (tobacco) in vivo using biolistic transformation. Surprisingly, it turned out that the spinach site is edited in the heterologous nuclear background. Furthermore, only exon-exon fusions were edited, whereas intron-containing messages remained unprocessed. A homologue of the spinach site was found to be present and edited in Nicotiana tomentosiformis, representing the paternal parent, but absent from Nicotiana sylvestris, representing the maternal parent of tobacco. Our data show that: (i) the cis-determinants for ndhA editing are split by an intron; (ii) the editing capacity cannot be deduced from editing sites; and (iii) allopolyploidization can increase the editing capacity, which implies that it can influence speciation processes in evolution.
Subject(s)
Chloroplasts/genetics , Chloroplasts/metabolism , NADH Dehydrogenase/genetics , Nicotiana/genetics , Plants, Toxic , RNA Editing , RNA Splicing , Spinacia oleracea/genetics , Base Sequence , Biolistics , Chromosome Mapping , Exons , Introns , Molecular Sequence Data , Plastids/genetics , Polyploidy , Sequence Alignment , Sequence Homology, Nucleic Acid , Spinacia oleracea/enzymology , Nicotiana/enzymologyABSTRACT
Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.
Subject(s)
Evolution, Molecular , Introns , Nuclear Proteins/physiology , Plant Proteins/physiology , RNA Splicing , RNA-Binding Proteins/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Chloroplasts , Cloning, Molecular , DNA, Plant , Genes, Plant , Molecular Sequence Data , Nuclear Proteins/genetics , Plant Proteins/genetics , Protein Biosynthesis , RNA Splicing Factors , RNA, Messenger , RNA-Binding Proteins/genetics , Rabbits , Repetitive Sequences, Nucleic Acid , Ribonucleoproteins/metabolism , Zea mays/geneticsABSTRACT
The chloroplast chromosome of spinach (Spinacia oleracea) is a double-stranded circular DNA molecule of 150,725 nucleotide pairs. A comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure. Three classes of open reading frames were distinguished: (1) genes of known function which include 108 unique loci, (2) three hypothetical chloroplast reading frames (ycfs) that are highly conserved interspecifically, and (3) species-specific or rapidly diverging 'open reading frames'. A detailed transcript study of one of the latter (ycf15) shows that these loci may be transcribed, but do not constitute protein-coding genes.
