Identification of a Chloroplast Ribonucleoprotein Complex ContainingTrans-splicing Factors, Intron RNA, and Novel Components
Molecular & Cellular Proteomics
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.
... ing 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. Molecular & Cellular Proteomics 1 The abbreviations used are: CRM, chloroplast RNA-splicing and ribosome maturation; EMSA, electrophoretic mobility shift assay; MudPIT, multidimensional protein identification technology; Ni-NTA, nickel-nitrilotriacetic acid; qRT-PCR, quantitative real-time PCR; SD, synthetic dropout; snRNA, small nuclear RNA; TAP, tandem affinity purification. Research