CRS1, a Chloroplast Group II Intron Splicing Factor, Promotes Intron Folding through Specific Interactions with Two Intron Domains (original) (raw)
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Proceedings of the National Academy of Sciences, 2009
Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring ''domain of unknown function 860'' (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 (''what's this factor?''), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions.
A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts
THE PLANT CELL ONLINE, 2007
Chloroplast genomes in land plants harbor ;20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1-and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.
Nuclearly Encoded Splicing Factors Implicated in RNA Splicing in Higher Plant Organelles
Molecular Plant, 2010
Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleoprotein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.
Group II intron splicing factors derived by diversification of an ancient RNA-binding domain
2003
Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identi®ed CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2±CAF1 and CRS2±CAF2 complexes have distinct intron speci®cities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNAbinding module that has diversi®ed to mediate speci®c interactions with various highly structured RNAs.
CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin
RNA, 2001
Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.
An mTERF domain protein functions in group II intron splicing in maize chloroplasts
Nucleic Acids Research, 2014
The mitochondrial transcription termination factor (mTERF) proteins are nucleic acid binding proteins characterized by degenerate helical repeats of 30 amino acids. Metazoan genomes encode a small family of mTERF proteins whose members influence mitochondrial gene expression and DNA replication. The mTERF family in higher plants consists of roughly 30 members, which localize to mitochondria or chloroplasts. Effects of several mTERF proteins on plant development and physiology have been described, but molecular functions of mTERF proteins in plants are unknown. We show that a maize mTERF protein, Zm-mTERF4, promotes the splicing of group II introns in chloroplasts. Zm-mTERF4 coimmunoprecipitates with many chloroplast introns and the splicing of some of these introns is disrupted even in hypomorphic Zm-mterf4 mutants. Furthermore, Zm-mTERF4 is found in high molecular weight complexes that include known chloroplast splicing factors. The splicing of two transfer RNAs (trnI-GAU and trnA-UGC) and one ribosomal protein messenger RNA (rpl2) is particularly sensitive to the loss of Zm-mTERF4, accounting for the loss of plastid ribosomes in Zm-mTERF4 mutants. These findings extend the known functional repertoire of the mTERF family to include group II intron splicing and suggest that a conserved role in chloroplast RNA splicing underlies the physiological defects described for mutations in BSM/Rugosa2, the Zm-mTERF4 ortholog in Arabidopsis.
Journal of Biological Chemistry, 2005
CRS2-associated factors 1 and 2 (CAF1 and CAF2) are closely related proteins that function in concert with chloroplast RNA splicing 2 (CRS2) to promote the splicing of specific sets of group II introns in maize chloroplasts. The CRS2-CAF complexes bind tightly to their cognate group II introns in vivo, with the CAF subunit determining the intron specificity of the complex. In this work we show that the CRS2-CAF complexes are stable in the absence of their intron targets and that CRS2 binds a 22 amino acid motif in the COOH-terminal region of CAF2 that is conserved in CAF1. Yeast two-hybrid assays and co-fractionation studies using recombinant proteins show that this motif is both necessary and sufficient to bind CRS2. The 22-amino acid motif is predicted to form an amphipathic helix whose hydrophobic surface is conserved between CAF1 and CAF2. We propose that this surface binds the hydrophobic patch on the surface of CRS2 previously shown to be necessary for the interaction between CRS2 and CAF2. Group II introns are large, catalytic RNAs defined by a conserved structural organization consisting of six largely helical domains, characteristic interdomain interactions and several limited regions of conserved sequence (1-4). A few group II introns have been shown to self-splice in vitro; however, to do so typically requires non-physiological conditions. In vivo, proteins are required for efficient splicing, presumably to aid the folding of these large, intricately structured RNAs into their catalytically competent conformations (5, 6). Group II introns are found in bacteria, mitochondria, and chloroplasts, and, sporadically, in archaea (6-9). The majority of bacterial group II introns are predicted to have the canonical group II intron fold and to encode a "maturase" protein that facilitates both splicing and intron mobility (7, 10). In contrast, many organellar group II introns deviate in their primary sequence and predicted structure from canonical self-splicing group II introns, do not encode a maturase protein, and fail to exhibit self-splicing activity. In particular, none of the ϳ40 group II introns found in the chloroplasts and mitochondria of higher plants have been observed to splice autonomously (11-13). The loss of selfsplicing activity and maturase open reading frames from organellar introns was accompanied by the recruitment of host-encoded protein cofactors that are essential for splicing (5, 6, 10, 11).