Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components

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. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1, 2). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs).1 This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3), reminiscent of eukaryotic spliceosomal RNA (1). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5, 6). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5, 6). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5, 7). I...

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.…”

“…By using Raa4 as a bait, Rat2 was found among subcomplex I proteins (24). RT-PCR analysis, data from LC-MS analyses (24), and a new gene model prediction of the recent C. reinhardtii database version 5.5 led to an extension of the previously predicted size of the proposed gene product to about 144 kDa. A reevaluation of the gene annotation was conducted with mutant L135F, which is allelic to the previously described mutant TR72:75 (22) (supplemental Fig.…”

“…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.…”

“…Three biological replicates of RT2T were applied to TAP and purified proteins were identified using multidimensional protein identification technology (MudPIT) (24). As a control to identify false positive proteins, we used strain RST-1, which carries a fusion of the RBCS1 gene with the TAP tag (24).…”

“…As a control to identify false positive proteins, we used strain RST-1, which carries a fusion of the RBCS1 gene with the TAP tag (24). In total, 20 proteins were identified in all three RT2T replicates (P1-P3, supplemental Table S1).…”