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

Abstract

SUMMARYSplicing 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.

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

“…Recent results revealed that splicing of psaA-i1 and -i2 is assisted by at least two high molecular weight protein complexes (subcomplexes I and II) harboring the general splicing factor Raa1 together with intron-specific subunits (17,24). Here, we provide the first evidence for a high molecular weight apparatus involved in trans-splicing of the psaA mRNA, which is the first report of a high molecular weight splicing apparatus from a eukaryotic organelle.…”

“…A total of four independent TAP experiments were performed. Proteins were considered as specific if they were found in four separate biological replicates and not in control experiments (C1-C4) (17). These analyses revealed 19 putative Raa2-interacting proteins (supplemental Table S2).…”

“…Previous studies have led to the identification of two transsplicing subcomplexes (I and II) comprising described factors and several unknown proteins that are involved in the splicing of both chloroplast group II introns (17,24). We aimed to elucidate core components of both subcomplexes.…”

“…Raa2 was already found in TAP tag experiments with trans-splicing factor Raa7 as bait (17). Using mutant A18, which lacks a functional Raa2 protein, as recipient, we have constructed strain R2T, which generates a Raa2::TAP fusion.…”

“…For C. reinhardtii, seven splicing factors, specific for group II introns, have been described at the molecular level (17)(18)(19)(20)(21)(22)(23). Whereas splicing factor Raa1 (RNA maturation of psaA RNA) is involved in splicing of both reactions, Raa3, Raa4, Raa8, and Rat2 (RNA maturation of psaA tscA RNA) are psaA-i1-specific.…”