Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces

Chloroplast RNA splicing 2 (CRS2) is a nuclear-encoded protein required for the splicing of nine group II introns in maize chloroplasts. CRS2 functions in the context of splicing complexes that include one of two CRS2-associated factors (CAF1 and CAF2). The CRS2-CAF1 and CRS2-CAF2 complexes are required for the splicing of different subsets of CRS2-dependent introns, and they bind tightly and specifically to their genetically defined intron targets in vivo. The CRS2 amino acid sequence is closely related to those of bacterial peptidyl-tRNA hydrolases (PTHs). To identify the structural differences between CRS2 and bacterial PTHs responsible for CRS2's gains of CAF binding and intron splicing functions, we determined the structure of CRS2 by X-ray crystallography. The fold of CRS2 is the same as that of Escherichia coli PTH, but CRS2 has two surfaces that differ from the corresponding surfaces in PTH. One of these is more hydrophobic in CRS2 than in PTH. Site-directed mutagenesis of this surface blocked CRS2-CAF complex formation, indicating that it is the CAF binding site. The CRS2 surface corresponding to the putative tRNA binding face of PTH is considerably more basic than in PTH, suggesting that CRS2 interacts with group II intron substrates via this surface. Both the sequence and the structural context of the amino acid residues essential for peptidyl-tRNA hydrolase activity are conserved in CRS2, yet expression of CRS2 is incapable of rescuing a pth(ts)E.coli strain.     

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain

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 identified 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 specificities. 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 RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.     

Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts

Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.     

A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts

Chloroplast genomes in land plants harbor approximately 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.     

Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.     

CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.     

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.     

Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts

Group II introns are catalytic RNAs that have been proposed to be the evolutionary precursors to the spliceosome. Most group II introns require accessory factors to splice efficiently in vivo, but few such factors have been identified. We have cloned the maize nuclear gene crs2, which is required for the splicing of nine group II introns in chloroplasts. CRS2 is related to peptidyl-tRNA hydrolase enzymes. However, CRS2 expression failed to rescue an Escherichia coli pth(ts) mutant and CRS2 lacks several conserved amino acids that are important for the activity of the E.coli enzyme, indicating that it may lack peptidyl-tRNA hydrolase activity. CRS2 is localized to the chloroplast stroma, where it is found in a large salt-stable complex that contains RNA. CRS2 co-sediments with group II intron RNA during centrifugation of stroma through sucrose gradients, suggesting that CRS2 facilitates splicing via direct interaction with intron RNA. Sequence comparisons indicate how evolutionary tinkering may have allowed an enzyme that interacts with peptidyl-tRNAs to acquire a function in group II intron splicing.     

Mutational analysis of functional domains in Mrs2p, the mitochondrial Mg2+ channel protein of Saccharomyces cerevisiae

The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants. Characteristic features of the Mrs2 protein family and the distantly related CorA proteins of bacteria are the presence of two adjacent transmembrane domains near the C terminus of Mrs2p one of which ends with a F/Y-G-M-N motif. Two coiled-coil domains and several conserved primary sequence blocks in the central part of Mrs2p are identified here as additional characteristics of the Mrs2p family. Gain-of-function mutations obtained upon random mutagenesis map to these conserved sequence blocks. They lead to moderate increases in mitochondrial Mg2+ concentrations and concomitant positive effects on splicing of mutant group II intron RNA. Site-directed mutations in several conserved sequences reduce Mrs2p-mediated Mg2+ uptake. Mutants with strong effects on mitochondrial Mg2+ concentrations also have decreased group II intron splicing. Deletion of a nonconserved basic region, previously invoked for interaction with mitochondrial introns, lowers intramitochondrial Mg2+ levels as well as group II intron splicing. Data presented support the notion that effects of mutations in Mrs2p on group II intron splicing are a consequence of changes in steady-state mitochondrial Mg2+ concentrations.     

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.     

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin

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.     

APO1 promotes the splicing of chloroplast group II introns and harbors a plant-specific zinc-dependent RNA binding domain

Arabidopsis thaliana APO1 is required for the accumulation of the chloroplast photosystem I and NADH dehydrogenase complexes and had been proposed to facilitate the incorporation of [4Fe-4S] clusters into these complexes. The identification of maize (Zea mays) APO1 in coimmunoprecipitates with a protein involved in chloroplast RNA splicing prompted us to investigate a role for APO1 in splicing. We show here that APO1 promotes the splicing of several chloroplast group II introns: in Arabidopsis apo1 mutants, ycf3-intron 2 remains completely unspliced, petD intron splicing is strongly reduced, and the splicing of several other introns is compromised. These splicing defects can account for the loss of photosynthetic complexes in apo1 mutants. Recombinant APO1 from both maize and Arabidopsis binds RNA with high affinity in vitro, demonstrating that DUF794, the domain of unknown function that makes up almost the entirety of APO1, is an RNA binding domain. We provide evidence that DUF794 harbors two motifs that resemble zinc fingers, that these bind zinc, and that they are essential for APO1 function. DUF794 is found in a plant-specific protein family whose members are all predicted to localize to mitochondria or chloroplasts. Thus, DUF794 adds a new example to the repertoire of plant-specific RNA binding domains that emerged as a product of nuclear-organellar coevolution.     

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.     

Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16

Saccharomyces cerevisiae Prp22 and Prp16 are RNA-dependent ATPases required for pre-mRNA splicing. Both proteins are members of the DEXH-box family of nucleic acid-dependent NTPases. Prior mutational analysis of Prp22 and Prp16 identified residues within conserved motifs I (GXGKT), II (DEAH), and VI (QRXGRXGR) that are required for their biological activity. Nonfunctional Prp22 and Prp16 mutants exerted a dominant negative effect on cell growth. Here we show that overexpression of lethal Prp22 mutants leads to accumulation of unspliced pre-mRNAs and excised introns in vivo. The biochemical basis for the lethality and inhibition of splicing in vivo was determined by purifying and characterizing recombinant mutant proteins. The lethal Prp22 mutants D603A and E604A in motif II and Q804A and R808A in motif VI were defective for ATP hydrolysis and mRNA release from the spliceosome, but were active in promoting step 2 transesterification. Lethal Prp16 mutants G378A and K379A in motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry. The ATPase-defective mutants of Prp16 and Prp22 bound to spliceosomes in vitro and blocked the function of the respective wild-type proteins in trans. Comparing the mutational effects in Prp16 and Prp22 highlights common as well as distinct structural requirements for the ATP-dependent steps in pre-mRNA splicing.