RESUMO
During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.
Assuntos
Chlamydomonas reinhardtii/genética , Proteínas de Cloroplastos/metabolismo , Íntrons , Proteínas de Plantas/metabolismo , RNA de Cloroplastos/genética , Trans-Splicing , Proteínas de Cloroplastos/genética , Teste de Complementação Genética , Mutação , Conformação de Ácido Nucleico , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Group II introns with highly complex RNA structures have been discovered in both prokaryotes and eukaryotic organelles. Usually, excision of non-coding group II intron sequences occurs by cis-splicing, the intramolecular ligation of exons in the same precursor RNA, but some group II introns are excised by intermolecular ligation. This process is called trans-splicing, and genome sequencing predicted that this type of RNA processing occurs in more than 180 organelle genomes from eukaryotes. A well characterised trans-spliced intron RNA is represented by the chloroplast psaA gene of the model alga Chlamydomonas reinhardtii. The psaA gene is split into three exons, which are widely distributed over the plastome and transcribed independently. PsaA exons are flanked by sequences typical for group II introns and joined by trans-splicing via two transesterification reactions. Although it is known that some group II introns are able to splice autocatalytically, trans-splicing of the psaA RNA depends on several nucleus and chloroplast encoded factors. The phylogenetic relationship between group II introns and nuclear spliceosomal RNA led to the hypothesis that these factors are part of large multiprotein and ribonucleoprotein complexes akin to the nuclear spliceosome. Here, we give a concise overview of experimental strategies to identify novel factors involved in trans-splicing of psaA RNA and review recent results that have elucidated the composition and function of a putative chloroplast spliceosome involved in processing of chloroplast precursor RNAs.
Assuntos
Cloroplastos/metabolismo , Spliceossomos/metabolismo , Trans-Splicing/genética , Chlamydomonas reinhardtii/metabolismoRESUMO
In eukaryotes, RNA trans-splicing is an important RNA-processing form for the end-to-end ligation of primary transcripts that are derived from separately transcribed exons. So far, three different categories of RNA trans-splicing have been found in organisms as diverse as algae to man. Here, we review one of these categories: the trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. Trans-spliced exons can be predicted from DNA sequences derived from a large number of sequenced organelle genomes. Further molecular genetic analysis of mutants has unravelled proteins, some of which being part of high-molecular-weight complexes that promote the splicing process. Based on data derived from the alga Chlamydomonas reinhardtii, a model is provided which defines the composition of an organelle spliceosome. This will have a general relevance for understanding the function of RNA-processing machineries in eukaryotic organelles.
Assuntos
Cloroplastos/metabolismo , Íntrons , Mitocôndrias/metabolismo , RNA/genética , Trans-Splicing , Animais , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Humanos , Mitocôndrias/genética , Conformação de Ácido Nucleico , RNA/química , Spliceossomos/genéticaRESUMO
In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.
Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Íntrons , Proteínas de Ligação a RNA/metabolismo , Proteínas de Algas/classificação , Proteínas de Algas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Dados de Sequência Molecular , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , RNA de Algas/química , RNA de Algas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
In photoautotrophic organisms, the expression of nuclear genes encoding plastid proteins is known to be regulated at various levels. In this study, we present the analysis of two non-photosynthetic mutants (CC1051 and TR72) from the unicellular green alga Chlamydomonas reinhardtii. Both mutant strains show a defect in the processing of chloroplast psaA mRNA, and therefore they are assumed to be defective in photosystem I (PSI) assembly. We have performed macroarray experiments with trans-splicing mutants CC1051 and TR72 in order to analyse putative pleiotropic effects of nuclear-located mutations leading to a non-functional PSI. To the best of our knowledge, this is the first example of Chlamydomonas cDNA macroarray analysis comparing the transcriptional regulation of nuclear genes in wild-type and photosystem I mutants. The macroarray results demonstrated a transcriptional downregulation of members of the Lhcb gene family more than 2-fold in both mutant strains. In addition, real-time RT-PCR experiments found a 4- to 16-fold reduction in transcript levels of several Lhca genes in TR72; whereas in CC1051, no significant change in transcript levels was observed. Taken together, our data suggest that a signal is transmitted from the chloroplast to the nucleus that serves to regulate the level of light harvesting polypeptides in the organelle.
