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Plant J. 2016 Jan;85(1):57-69. doi: 10.1111/tpj.13089.

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii.

Author information

1
Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211, Geneva 4, Switzerland.
2
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr University Bochum, Universitätsstraße 150, Bochum, 44801, Germany.
3
Leibniz-Institut für Analytische Wissenschaften- ISAS - e.V., Otto Hahn Straße 6b, Dortmund, 44227, Germany.
4
Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, UK.
5
Medizinische Fakultät, Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstraße 150, Bochum, 44801, Germany.
6
Department of Analytical Chemistry, Ruhr-University Bochum, Universitätsstraße 150, Bochum, 44801, Germany.

Abstract

Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity.

KEYWORDS:

Chlamydomonas reinhardtii; chloroplast; plastid; psaA; splicing complex; tandem affinity purification; trans-splicing

PMID:
26611495
DOI:
10.1111/tpj.13089
[Indexed for MEDLINE]
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