RESUMO
With CR-RT-PCR as primary approach we mapped the 5' and 3' transcript ends of all mitochondrial protein-coding genes in Arabidopsis thaliana. Almost all transcripts analyzed have single major 3' termini, while multiple 5' ends were found for several genes. Some of the identified 5' ends map within promoter motifs suggesting these ends to be derived from transcription initiation while the majority of the 5' termini seems to be generated post-transcriptionally. Assignment of the extremities of 5' leader RNAs revealed clear evidence for an endonucleolytic generation of the major cox1 and atp9 5' mRNA ends. tRNA-like structures, so-called t-elements, are associated either with 5' or with 3' termini of several mRNAs. These secondary structures most likely act as cis-signals for endonucleolytic cleavages by RNase Z and/or RNase P. Since no conserved sequence motif is evident at post-transcriptionally derived ends, we suggest t-elements, stem-loops and probably complex higher order structures as cis-elements for processing. This analysis provides novel insights into 5' and 3' end formation of mRNAs. In addition, the complete transcript map is a substantial and important basis for future studies of gene expression in mitochondria of higher plants.
Assuntos
Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA de Plantas/química , RNA/química , Arabidopsis/enzimologia , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/metabolismo , Processamento de Terminações 3' de RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA de Plantas/metabolismo , RNA de Transferência/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5' sequences up to nucleotide position -584, the coding regions and the 3' flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position -584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5' end is abundant. This terminus and the other major 5' ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5' terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5' mRNA end.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Mitocondriais/genética , RNA Mensageiro/química , Região 5'-Flanqueadora , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Sequência de Bases , Southern Blotting , Núcleo Celular/genética , DNA Mitocondrial/análise , DNA Mitocondrial/química , DNA Mitocondrial/genética , Ordem dos Genes , Variação Genética , Proteínas Mitocondriais/biossíntese , ATPases Mitocondriais Próton-Translocadoras/genética , RNA Mensageiro/análise , Sequências Repetitivas de Ácido NucleicoRESUMO
In our analysis of 5' and 3' end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5' termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F(1) hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5' end generation of mitochondrial transcripts.