RNA editing in higher plant plastids: oligoribonucleotide SSCP analysis allows the proof of base conversion directly at the RNA level.

Plastid RNA editing of a number of transcripts at specific sites changes genomically encoded cytidines to nucleosides, which act like uridines in RT-PCR analyses. To study plastid-editing directly at the RNA level, we established a single-strand conformational polymorphism assay for the discrimination of small RNA molecules. The electrophoretic mobility of a oligoribonucleotide resulting from a RNase T1-digested and edited plastid mRNA was shown to be identical with a control RNA molecule containing a uridine at the editing site, whereas the unedited RNA behaved like a RNA molecule containing a cytidine at the respective position.

Constitutive expression of interferon-induced human MxA protein in transgenic tobacco plants does not confer resistance to a variety of RNA viruses.

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses, including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35 S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplication of tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.

Transcriptionally active chromosomes (TACs) of barley chloroplasts contain the alpha-subunit of plastome-encoded RNA polymerase.

Transcriptionally active chromosomes (TACs) were isolated from mature chloroplasts of barley, from proplastids enriched in basal segments of barley primary foliage leaves, and from ribosome-deficient plastids of heat-bleached barley leaves. Immunological analysis with a specific antibody raised against the plastid rpoA gene product revealed that chloroplasts contain an immunoreactive protein of 38 kDa in the TAC fraction which appears to be identical to the alpha-subunit contained in the soluble RNA polymerase (sRNAP) fraction of the same chloroplasts. However, only traces of immunoreactive protein were detected in a TAC preparation derived from "proplastids". A positive correlation could be demonstrated between transcriptional activity and the amount of immunoreactive 38-kDa protein by analyzing different TAC fractions eluting at different times during gel filtration of a standard TAC preparation as well as in TAC preparations obtained under various detergent conditions.

RNA editing in plant mitochondria and chloroplasts.

In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs. The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.

A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited.

The RNA editing processes in chloroplasts and mitochondira of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. An rpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrial rpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potential rpoB editing sites previously detected in maize were found to be edited in the rice chloroplast rpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

Analysis of transcript accumulation and splicing in plastids of four nuclear mutants of barley revealed that the ribosomal protein L2 (rpl2) gene transcripts containing a group II intron remained entirely unspliced, whereas the intron of the ribosomal protein L16 (rpl16) gene (linked with the rpl2 gene in the same operon) was removed in the mutant plastids. Also, the transcripts of other genes containing group II introns (ribosomal protein S16 gene, rps16; NADH dehydrogenase ND2 gene, ndhB; cytochrome f gene, petD; and intron-containing reading frame 170, irf170) and of the tRNA for leucine, trnL (UAA), possessing the only chloroplast group I intron, were found to be spliced. The mutants used in this investigation are considered to be nonallelic; this excludes the possibility that a single nuclear gene is responsible for the impaired splicing of rpl2 transcripts. The mutants, however, have a severe deficiency in chloroplast ribosomes in common; this deficiency is evident from the lack of the essential ribosomal protein L2 and from an extremely low steady state level of plastid rRNAs. From these results, we conclude that a functioning translational apparatus of the organelle is a prerequisite for splicing of the chloroplast rpl2 class II intron but not for splicing of at least five other group II intron-containing transcripts. This provides genetic evidence for a chloroplast DNA-encoded component (e.g., a maturase) involved in the splicing of rpl2 pre-mRNA.

The role of RNA editing in conservation of start codons in chloroplast genomes.

Open reading frames (ORFs), encoded by the plastid genomes of tobacco, liverwort, rice and maize were aligned with a view to studying the conservation of translational start and stop codons created by RNA editing of homologous genes. It became evident that most of the homologous ORFs have conserved translation start and stop signals at the gene level. However, some of the ORFs show differences with respect to extensions of their 3' and 5' terminal regions. For example, the proposed N-termini of the ndhD-encoded peptides from different plant species are very variable in length and amino-acid composition. Sequence analysis of ndhD and the corresponding cDNA shows that editing of an ACG triplet in tobacco, spinach and snapdragon leads to the creation of an AUG codon, corresponding to the start codon in other species. Conservation of translational start codons of plastome-encoded genes can, therefore, be achieved by editing of transcripts, and the definition of plastome-encoded ORFs must take potential editing events into consideration.

Complete RNA editing of unspliced and dicistronic transcripts of the intron-containing reading frame IRF170 from maize chloroplasts.

The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA.

Editing of the chloroplast rpoB transcript is independent of chloroplast translation and shows different patterns in barley and maize.

Sequence analysis of amplified cDNAs derived from the maize chloroplast rpoB transcript which encodes the beta subunit of a chloroplast specific, DNA dependent RNA polymerase reveals four C-to-U editing sites clustered within 150 nucleotides of the 5' terminal region of the rpoB message. These newly identified editing sites confirm the bias of chloroplast editing for certain codon transitions and for second codon positions which both appear suggestive for an involvement of the translational apparatus in the editing process. This supposition prompted us to investigate editing of the rpoB transcript from ribosome deficient, and hence protein synthesis deficient, plastids of the barley mutant albostrians. In this mutant editing is, however, not impaired at any of the editing sites functional in the barley wild type rpoB transcript. This demonstrates that chloroplast editing is neither linked to nor dependent on the chloroplast translational apparatus. As a further consequence any peptide components required for chloroplast editing must be encoded in the nuclear genome. In spite of strong sequence conservation only three of the four editing sites identified in the maize rpoB transcript are functional in barley. This indicates that sequences surrounding an editing site alone are not sufficient as determinants for the editing process in chloroplasts, but that trans-acting templates carrying the editing information for each individual site may also be required.

Internal editing of the maize chloroplast ndhA transcript restores codons for conserved amino acids.

The NADH dehydrogenase subunit A (ndhA) gene from maize chloroplasts encodes a highly conserved peptide, which at several positions could be restored to consensus sequences by potential C-to-U editing of the codons involved. This gene was, therefore, chosen for analysis of its mRNA sequence in the form of amplified cDNA. A comparison of this cDNA sequence with the plastome-encoded ndhA sequence reveals four C-to-U editing sites, thereby demonstrating as a novel finding that chloroplast editing can also affect internal mRNA positions. All the edited codons restore amino acids that are conserved in the ndhA-encoded peptides of other chloroplast species. Alignment with homologous mitochondrial NADH-ubiquinone reductase subunit 1 (nad1) sequences of plant and even nonplant species shows that two of the editing positions restore universally conserved amino acids and that one editing site is even shared with nad1 mRNA of plant mitochondria. No editing sites could be detected in the cDNA derived from transcripts of the maize chloroplast RNA polymerase alpha-subunit (rpoA) gene.

Alternative base pairing between 5'- and 3'-terminal sequences of small subunit RNA may provide the basis of a conformational switch of the small ribosomal subunit.

The compiled sequences of small subunit ribosomal RNAs have been screened for base complementary between 5'- and 3'-terminal regions. Highly conserved complementary sequences are found which allow formation of a helix between the two ends of 5 or 6 base pairs. This helix is composed of sequences from the loop region of the first 5'-terminal stem and from sequences immediately distal to the last stem (the Me2A-stem) of the 3' terminus and therefore allows a coaxial stacking with either of these two flanking stems. Formation of the 5'/3'-helical arrangement is, however, only possible at the cost of dissolving the 'pseudo-knot' helix between the 5'-terminal region and the internal region of small subunit RNA. It is postulated that the mutually exclusive conformational states are in dynamic equilibrium and that they correlate with distinct functional states of the small ribosomal subunit. The 'pseudo-knot' containing conformation with the 3'-terminal sequences more exposed is likely to represent the initiating state, whereas the 5'/3' terminal paired 'closed' conformation may represent the elongating state in which interaction with fortuitous ribosomal binding sequences of mRNAs is avoided.