A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.     

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.     

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.     

The pentatricopeptide repeat protein EMB1270 interacts with CFM2 to splice specific group II introns in Arabidopsis chloroplasts

Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes. Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat (PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana. Knockout of EMB1270 led to embryo arrest, whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I (PSI) subunits were significantly reduced, whereas the levels of photosystem II (PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2, ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay (REMSA), a truncated EMB1270 protein containing the 11 N-terminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns. In addition, EMB1270 specifically interacted with CRM Family Member 2 (CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.     

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.     

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.     

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.     

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 DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize(Zea mays) DEAD-box RNA helicase48(Zm RH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis,and seed development. Loss of Z...     

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.     

A PORR domain protein required for rpl2 and ccmF(C) intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria

Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus‐encoded splicing factors have been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified: rpl2 and ccmF_C. WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in wtf9 mutants as a means to functionally characterize the mitochondrial rpl2 and ccmF_C genes. Loss of ccmF_C expression correlates with the loss of cytochromes c and c₁, confirming a role for ccmF_C in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial rpl2 gene, and imply that the Rpl2 fragment encoded by rpl2 exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein in trans.