A single nuclear locus is involved in both chloroplast RNAtrans‐splicing and 3′ end processing

Hahn, Daniela; Nickelsen, Jörg; Hackert, Anke; Kück, Ulrich

doi:10.1046/j.1365-313x.1998.00234.x

Cited by 31 publications

(37 citation statements)

References 30 publications

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“…Northern analysis confirmed that mutant F19 has a defect in trans splicing of the first group II intron but not in 3=-end processing of tscA RNA. We previously showed that trans-splicing mutants with a defect in tscA processing generated precursor RNA molecules of 1,700 nt and 2,800 nt that are clearly distinguished from the processed, 450-nt tscA RNA (15,37). All analyzed complementation strains exhibited restored photosystem I activity and regained the ability to generate mature psaA mRNA.…”

Section: Resultsmentioning

confidence: 92%

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

2015

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The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors. Interaction modules are crucial for the molecular and cellular function of proteins, and a wide repertoire of diverse domains and motifs mediates a variety of regulatory mechanisms. Proteins that exhibit a repeating structural unit with an ␣-helical architecture are classified as members of the ␣-solenoid superfamily (1). This large family is widely distributed in eukaryotes and includes, among others, the transcription activator-like (TAL) effectors, the tetratricopeptide repeat (TPR) proteins, and the pentatricopeptide repeat (PPR) proteins (2-4). The ␣-helical structure of diverse members mediates binding to either proteins or nucleic acids.A well-characterized example of RNA binding proteins is the PPR protein family (5). The degenerate PPR motif is described as two antiparallel, ␣-helical tracts of 35 amino acids, whose repeats are often arranged in tandem arrays. Recently, two approaches provided insights into the predictable RNA binding code of this motif (6, 7). Structural analyses indicate that RNA binding is mediated by helical tracts forming a superhelical groove with exposed residues. Despite the low sequence conservation of the PPR motif being restricted to several amino acids, PPR proteins specifically recognize single-stranded RNA sequences through a combinatorial amino acid code (2, 6, 7). Mod...

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Section: Resultsmentioning

confidence: 92%

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

2015

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“…In the case of the class B mutant HN31, the factor affected by the mutation appears to be a bifunctional protein. It is involved both in the maturation of the tscA RNA required for splicing of exons 1 and 2, and also in the second transsplicing reaction (Hahn et al 1998). …”

Section: Discussionmentioning

confidence: 99%

A multiprotein complex involved in chloroplast group II intron splicing

Perron

Goldschmidt-Clermont²,

Rochaix³

2004

RNA

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The psaA gene of the green alga Chlamydomonas reinhardtii consists of three exons that are widely separated on the chloroplast genome and transcribed independently. The exons are flanked by group II intron sequences. Maturation of the psaA mRNA requires two steps of splicing in trans between the transcripts of exons 1, 2, and 3. At least 14 nuclear loci and one chloroplast gene (tscA) are involved in this process. Recently the genes of three of these nuclear factors have been cloned. Raa3 is involved in the first trans-splicing reaction, and Raa1 and Raa2 are required for the second trans-splicing reaction. Here we show that Raa1 and Raa2 can be coimmunoprecipitated and that they are part of a high molecular weight complex of 400-500 kD. The size and integrity of the complex are affected by mutations in other complementation groups, suggesting that the corresponding proteins may also be components of this multiprotein complex or required for its assembly. Raa1 is also associated with a larger complex.

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“…5): class A mutants fail to trans-splice exon 2 and 3 primary transcripts; class B mutants neither splice exon 1 and 2 nor exon 2 and 3 primary transcripts; and class C mutants are not able to splice exon 1 and 2 primary transcripts. (85,86) Lack of correct splicing in class B and C mutants can take place at two different levels of RNA processing, i.e. either splicing of the primary psaA transcripts or 3 0 -end processing of the tscA RNA is affected.…”

Section: Ppr Proteinmentioning

confidence: 99%

Trans‐splicing of organelle introns – a detour to continuous RNAs

Glanz

Kück

2009

BioEssays

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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.

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A single nuclear locus is involved in both chloroplast RNAtrans‐splicing and 3′ end processing

Cited by 31 publications

References 30 publications

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

A multiprotein complex involved in chloroplast group II intron splicing

Trans‐splicing of organelle introns – a detour to continuous RNAs

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