The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis (original) (raw)
Related papers
Self-Splicing of the Chlamydomonas Chloroplast psbA Introns
THE PLANT CELL ONLINE, 1991
We used &'P-GTP labeling of total RNA preparations to identify self-splicing group I introns in Chlamydomonas. Several RNAs become labeled with CX-~~P-GTP, a subset of which is not seen with RNA from a mutant that lacks both copies of the psbA gene. Hybridization of the GTP-labeled RNAs to chloroplast DNA indicates that they originate from the psbA and rrn 23s genes, respectively, the only genes known to contain group I introns in this organism. lntrons 1, 2, and 3 of psbA (with flanking exon sequences) were subcloned and transcribed in vitro. The synthetic RNAs were found to self-splice; splicing required Mg2+, GTP, and elevated temperature. In addition, the accuracy of self-splicing was confirmed for introns 1 and 2, and intermediates in the splicing reactions were detected. These results, together with our recent data on the 23s intron, indicate that the ability to self-splice is a general feature of Chlamydomonas group I introns. These findings have significant implications for the mechanism of group I intron splicing and evolution in Chlamydomonas and other chloroplast genomes.
Nucleic Acids Research, 1989
We report the nucleotide sequence of the chloroplast psbA gene encoding the 32 kilodalton protein of photosystem II from Chlamydomonas moewusii. Like its land plant homologues, this green algal protein consists of 353 amino acids. The C. moewusii psbA gene is composed of three exons containing 252, 11 and 90 codons and of two group I introns containing 2363 and 1807 nucleotides. Each of the introns features an internal open reading frame (ORF) that potentially encodes a basic protein of more than 300 residues. The primary sequences of the putative intron-encoded proteins are unrelated and none of them shares conserved elements with any of the proteins predicted from the group I intron sequences published so far. The fist C. moewusii intron is inserted at the same position as the fourth intron of the psbA gene from Chlamydomonas reinhardtii; the second intron lies at a novel site downstream of this position. On the basis of their RNA secondary structures, the C. moewusii introns 1 and 2 can be assigned to subgroups IA and IB, respectively. However, intron 1 is not typical of subgroup IA introns, its most unusual feature being the location of the ORF in the "loop L5" region. To our knowledge, this is the first time that an ORF is located in this region of the group I intron structure.
The Plant Journal, 2002
Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU selfsplicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg 2 concentration. The mutant constructs were transformed into chloroplasts to replace the wild-type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-de®cient P4 125 A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which de®nes the chloroplast-splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild-type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast. Abbreviations: Chlamydomonas reinhardtii, C. reinhardtii; Cr.LSU, chloroplast 23S large subunit rRNA intron of C. reinhardtii; Cr.psbA4, fourth intron of the psbA gene in Chlamydomonas reinhardtii; Tt.LSU, cytoplasmic large subunit rRNA intron of Tetrahymena thermophila; T4.td, thymidylate synthase intron of phage T4; WT, wild type.
Molecular Biology and Evolution, 2013
Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells. Here, we provide in vivo evidence for reverse transcription of the chloroplast psbA mRNA in two naturally occurring species of Chlamydomonas (raudensis and subcaudata) that is based on the presence of spliced cDNAs in both organisms. The psbA cDNAs, which lack the group II intron of the genomic gene, are nearly full length, and the majority of themthough not all-are in the form of RNA-cDNA hybrids. Moreover, the presence in these species of psbA cDNAs is correlated with the loss of an early group I intron from the same psbA gene. The group II intron that interrupts psbA in C. raudensis and C. subcaudata potentially encodes a protein with a reverse transcriptase domain, and the C. raudensis protein was shown to have reverse transcriptase activity in vitro. These results provide strong evidence for reverse transcription of a physiologically important mRNA (psbA) in two species of Chlamydomonas that have also lost an intron from the same gene, possibly through recombination with the cDNA.
RNA, 2004
The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg 2+ (>12 mM) and NH 4 +. Selfsplicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ∼44°C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50°C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.
A small chloroplast RNA may be required for trans-splicing in chlamydomonas reinhardtii
Cell, 1991
In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans.splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans.splicing of exons I and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans-splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts.