Abstract
Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.
Highlights
Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features
Purification of Raa4 Complexes Using a Codon-optimized tandem affinity purification (TAP) Tag Reveals Novel Protein–Protein Interactions between Chloroplast Splicing Factors—To elucidate the protein interaction network that mediates psaA splicing, we selected the recently identified protein Raa4, which is involved in transsplicing of the first psaA intron [14], as a target protein in protein–protein interaction studies
For the purification of native Raa4 complexes, a vector was generated in which a codon-optimized TAP tag consisting of protein A, a tobacco etch virus protease cleavage site, and a calmodulin-binding peptide was C-terminally fused to Raa4
Summary
Conditions, and Transformation—C. reinhardtii strains and growth conditions are listed in supplemental Table S1. Rab yeast-two hybrid vectors were generated as follows: DNA fragments coding for amino acids 51–725 and 668 –1216 were amplified from cDNA (primers: OVK48, OVK49; OVK50, OVK51) and cloned into EcoRI and BamHI restriction sites of pGADT7 resulting in pGADT7_Rab1-A and pGADT7_Rab1-B. For the generation of two-hybrid vectors carrying Raa subfragments, RAA3 fragments coding for amino acids 674 –1298, 70 – 675, 1296 –1783, and 674 –1783 were amplified from cDNA (primers: for_ Raa, rev_Raa; for_Raa, rev_Raa; for_Raa, rev_ Raa; for_Raa, rev_Raa3-2) and inserted into EcoRI and SalI restriction sites of vector pGBKT7 resulting in plasmids pGBKT7_Raa3-A, pGBKT7_Raa3-B, pGBKT7_Raa3-C, and pGBKT7_. In order to construct RAT2 two-hybrid plasmids, cDNA was amplified in three fragments (primers: for_pGADT7_Rat, rev_Rat2_F1; for_Rat2_F2, rev_Rat2_F2; for_Rat2_F3, rev_pGADT7_Rat2) that overlapped each other and the pGADT7 cloning site. Detected proteins were considered as specific Raa binding partners if they were identified in all Raa4::TAP purification replicates but not in the negative controls (two TAP experiments with strain RST-1, expressing RBCS::TAP tag fusion gene, and two TAP experiments with the untagged wild type argϪcw15)
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