Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smalle... more Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral ...
Results of in vitro experiments have suggested the existence of at least three pathways by which ... more Results of in vitro experiments have suggested the existence of at least three pathways by which nuclearencoded proteins are targeted to the chloroplast thylakoid membrane. However, few components of the targeting machinery have been identified and the relationship between the three pathways is not clear. To investigate mechanisms underlying thylakoid protein targeting, we identified nuclear mutations in maize that cause targeting defects. We found two mutations, thal and hcflO6, that disrupt the localization of different sets of proteins to the thylakoid lumen. The thal mutation interferes with the targeting of one chloroplast-encoded protein, cytochrome f, and three nuclear-encoded proteins, plastocyanin, the psaF gene product and the 33 kDa subunit of the oxygen-evolving complex. The hcflO6 mutation interferes with the targeting of the 16 and 23 kDa subunits of the oxygenevolving complex. The thal and hcfiO6 phenotypes provide the first in vivo evidence supporting the existence of two distinct thylakoid-targeting pathways. Their phenotypes also provide evidence that one chloroplast-encoded protein, cytochrome f, engages the 'thal' pathway, indicating that nuclearand chloroplast-encoded proteins can be targeted via common machinery.
Methods in molecular biology (Clifton, N.J.), 2009
RNA-protein interactions profoundly impact organismal development and function through their cont... more RNA-protein interactions profoundly impact organismal development and function through their contributions to the basal gene expression machineries and their regulation of post-transcriptional processes. The repertoire of predicted RNA binding proteins (RBPs) in plants is particularly large, suggesting that the RNA-protein interactome in plants may be more complex and dynamic even than that in metazoa. To dissect RNA-protein interaction networks, it is necessary to identify the RNAs with which each RBP interacts and to determine how those interactions influence RNA fate and downstream processes. Identification of the native RNA ligands of RBPs remains a challenge, but several high-throughput methods for the analysis of RNAs that copurify with specific RBPs from cell extract have been reported recently. This chapter reviews approaches for defining the native RNA ligands of RBPs on a genome-wide scale and provides a protocol for a method that has been used to this end for RBPs that lo...
The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of transl... more The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames...
ABSTRACT During the evolution of chloroplasts from their bacterial ancestor traits emerged that a... more ABSTRACT During the evolution of chloroplasts from their bacterial ancestor traits emerged that are absent or rare in bacteria. Prominent among these acquired traits are RNA splicing and RNA editing. The numbers and distribution of introns and editing sites in different taxa suggest that editing and splicing have taken different evolutionary pathways in different chloroplast lineages. Both processes are dependent on nuclear-encoded factors and, intriguingly, PPR (pentatricopeptide repeat) proteins have recently been recognized as acommon player. This review summarizes recent progress in understanding the mechanisms, regulation, and trans-acting factors for these two types of RNA processing.
Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosom... more 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.
Plastids were derived through endosymbiosis from a cyanobacterial ancestor, whose uptake was foll... more Plastids were derived through endosymbiosis from a cyanobacterial ancestor, whose uptake was followed by massive gene transfer to the nucleus, resulting in the compact size and modest coding capacity of the extant plastid genome. Plastid gene expression is essential for plant development, but depends on nucleus-encoded proteins recruited from cyanobacterial or host-cell origins. The plastid genome is heavily transcribed from numerous promoters, giving posttranscriptional events a critical role in determining the quantity and sizes of accumulating RNA species. The major events reviewed here are RNA editing, which restores protein conservation or creates correct open reading frames by converting C residues to U, RNA splicing, which occurs both in cis and trans, and RNA cleavage, which relies on a variety of exoribonucleases and endoribonucleases. Because the RNases have little sequence specificity, they are collectively able to remove extraneous RNAs whose ends are not protected by RN...
The chloroplast is a compartment unique to photosynthetic eukaryotes. It and other members of the... more The chloroplast is a compartment unique to photosynthetic eukaryotes. It and other members of the plastid family carry out a remarkable range of activities. Explorations of photosynthesis-related phenomena have stimulated biochemical, biophysical, molecular and genetic approaches to understanding the proplastid-to-chloroplast conversion and the regulation of chloroplast processes. Much of this regulation is exerted by nuclear gene products, which often participate in plastid gene expression. Concerted exploitation of genomic resources promises a deeper understanding of these regulatory factors and more effective genetic engineering based on chloroplast transformation. Here, we review recent progress, drawing attention to maize, whose unique attributes and new resources hold special promise for explorations of chloroplast biogenesis. Glossary NPPs: nucleus-encoded plastid proteins.
The regulation of chloroplast translation by nuclear gene products makes a major contribution to ... more The regulation of chloroplast translation by nuclear gene products makes a major contribution to the control of chloroplast gene expression, but the underlying mechanisms are poorly understood. We describe a pentatricopeptide repeat (PPR) protein in maize, ATP4, that is necessary for translation of the chloroplast atpB open reading frame. We demonstrate that ATP4 associates in vivo with sequences near the 5' end of the unusually long 5' UTR of the atpB/E mRNA, that it facilitates ribosome association with this mRNA, and that it is required for accumulation and activity of the chloroplast ATP synthase. ATP4 is multifunctional, in that it also enhances atpA translation and is required for accumulation of specific processed atpF and psaJ transcripts. ATP4 belongs to a sub-class of PPR proteins that include a small MutS-related (SMR) domain. SMR domains had previously been associated primarily with DNA-related functions, but our findings imply that at least some PPR-SMR proteins can act on RNA. ATP4 is orthologous to the Arabidopsis protein SVR7, but the phenotypes of atp4 and svr7 mutants suggest that the functions of these orthologs have not been strictly conserved.
The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeating motif that is found in... more The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeating motif that is found in animal, fungal, and plant proteins. The PPR protein family is particularly large in plants, where the majority of family members are predicted to be targeted to mitochondria or chloroplasts. PPR proteins are believed to fall into the larger family of helical repeat proteins, which typically bind macromolecules through a surface formed by the stacking of consecutive helical repeating units. Prior findings implicate several PPR proteins in organellar RNA metabolism, but the biological functions of few PPR proteins have been explored and in no case has a direct substrate been definitively identified. We present a characterization of the maize nuclear gene ppr2, which encodes a chloroplast PPR protein. PPR2 is found in large, heterogeneous protein complexes in the chloroplast stroma, some of which may be associated with RNA. Null ppr2 mutants have albino leaves and lack plastid rRNA and translation products. Plastid rRNAs are absent in both dark- and light-grown leaf tissues, indicating that their absence does not result from photo-oxidative damage. The population of plastid transcripts in ppr2 mutants is similar to that in other maize mutants lacking plastid ribosomes, and no ppr2-specific defects in plastid RNA metabolism have been detected. Taken together, the results suggest that ppr2 functions in the synthesis or assembly of one or more component of the plastid translation machinery.
The pentatricopeptide repeat (PPR) is a degenerate 35–amino acid repeat motif that is widely dist... more The pentatricopeptide repeat (PPR) is a degenerate 35–amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown ...
Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as ... more 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...
The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNA... more The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNAs and for the processing of the petD mRNA from a polycistronic precursor. In order to understand the biochemical role of the crp1 gene product and the interconnections between chloroplast translation and RNA metabolism, the crp1 gene and cDNA were cloned. The predicted crp1 gene product (CRP1) is related to nuclear genes in fungi that play an analogous role in mitochondrial gene expression, suggesting an underlying mechanistic similarity. Analysis of double mutants that lack both chloroplast ribosomes and crp1 function indicated that CRP1 activates a site-specific endoribonuclease independently of any role it plays in translation. Antibodies prepared to recombinant CRP1 were used to demonstrate that CRP1 is localized to the chloroplast stroma and that it is a component of a multisubunit complex. The CRP1 complex is not associated detectably with either chloroplast membranes or chloroplast ribosomes. Models for CRP1 function and its relationship to other activators of organellar translation are discussed.
data). Because the CAFs and CRS1 have no significant similarity outside of their repeated domains... more data). Because the CAFs and CRS1 have no significant similarity outside of their repeated domains, it seems likely that these domains contribute to the splicing functions of these proteins, perhaps by binding RNA. The prokaryotic homologs of these domains have been
Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are ... more Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are related to the tetratricopeptide repeat (TPR). Most characterized PPR proteins mediate specific post-transcriptional steps in gene expression in mitochondria or chloroplasts. However, little is known about the structure of PPR proteins or the biochemical mechanisms through which they act. Here we establish features of PPR protein structure and nucleic acid binding activity through in vitro experiments with PPR5, which binds and stabilizes a chloroplast tRNA precursor harboring a group II intron. Recombinant PPR5 was shown to be monomeric by analytical ultracentrifugation and gel filtration. Circular dichroism spectroscopy showed that PPR5 has a high content of α helices, as predicted from the similarity between PPR and TPR motifs. PPR5 and another PPR protein, CRP1, bind with high affinity to single-stranded RNA, but bind poorly to single-stranded DNA or to double-stranded RNA or DNA. A ...
Proceedings of the National Academy of Sciences, 1991
We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon.... more We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and ...
Proceedings of the National Academy of Sciences, 2013
Significance Transcripts in plant organelles are altered by conversion of cytidines to uridines i... more Significance Transcripts in plant organelles are altered by conversion of cytidines to uridines in a process termed RNA editing. Members of two protein families have been identified in the plant editosome, but its complete composition is unknown. Now a unique protein that contains an RNA recognition motif has been found to be essential for editing of multiple plastid transcripts in both Arabidopsis and maize. Phylogenetic analysis indicates that this protein belongs to a sub-family of RNA recognition-motif proteins predominantly predicted to be targeted to organelles and that are thus likely to play roles in organelle RNA metabolism.
The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large fami... more The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.
Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smalle... more Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral ...
Results of in vitro experiments have suggested the existence of at least three pathways by which ... more Results of in vitro experiments have suggested the existence of at least three pathways by which nuclearencoded proteins are targeted to the chloroplast thylakoid membrane. However, few components of the targeting machinery have been identified and the relationship between the three pathways is not clear. To investigate mechanisms underlying thylakoid protein targeting, we identified nuclear mutations in maize that cause targeting defects. We found two mutations, thal and hcflO6, that disrupt the localization of different sets of proteins to the thylakoid lumen. The thal mutation interferes with the targeting of one chloroplast-encoded protein, cytochrome f, and three nuclear-encoded proteins, plastocyanin, the psaF gene product and the 33 kDa subunit of the oxygen-evolving complex. The hcflO6 mutation interferes with the targeting of the 16 and 23 kDa subunits of the oxygenevolving complex. The thal and hcfiO6 phenotypes provide the first in vivo evidence supporting the existence of two distinct thylakoid-targeting pathways. Their phenotypes also provide evidence that one chloroplast-encoded protein, cytochrome f, engages the 'thal' pathway, indicating that nuclearand chloroplast-encoded proteins can be targeted via common machinery.
Methods in molecular biology (Clifton, N.J.), 2009
RNA-protein interactions profoundly impact organismal development and function through their cont... more RNA-protein interactions profoundly impact organismal development and function through their contributions to the basal gene expression machineries and their regulation of post-transcriptional processes. The repertoire of predicted RNA binding proteins (RBPs) in plants is particularly large, suggesting that the RNA-protein interactome in plants may be more complex and dynamic even than that in metazoa. To dissect RNA-protein interaction networks, it is necessary to identify the RNAs with which each RBP interacts and to determine how those interactions influence RNA fate and downstream processes. Identification of the native RNA ligands of RBPs remains a challenge, but several high-throughput methods for the analysis of RNAs that copurify with specific RBPs from cell extract have been reported recently. This chapter reviews approaches for defining the native RNA ligands of RBPs on a genome-wide scale and provides a protocol for a method that has been used to this end for RBPs that lo...
The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of transl... more The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames...
ABSTRACT During the evolution of chloroplasts from their bacterial ancestor traits emerged that a... more ABSTRACT During the evolution of chloroplasts from their bacterial ancestor traits emerged that are absent or rare in bacteria. Prominent among these acquired traits are RNA splicing and RNA editing. The numbers and distribution of introns and editing sites in different taxa suggest that editing and splicing have taken different evolutionary pathways in different chloroplast lineages. Both processes are dependent on nuclear-encoded factors and, intriguingly, PPR (pentatricopeptide repeat) proteins have recently been recognized as acommon player. This review summarizes recent progress in understanding the mechanisms, regulation, and trans-acting factors for these two types of RNA processing.
Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosom... more 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.
Plastids were derived through endosymbiosis from a cyanobacterial ancestor, whose uptake was foll... more Plastids were derived through endosymbiosis from a cyanobacterial ancestor, whose uptake was followed by massive gene transfer to the nucleus, resulting in the compact size and modest coding capacity of the extant plastid genome. Plastid gene expression is essential for plant development, but depends on nucleus-encoded proteins recruited from cyanobacterial or host-cell origins. The plastid genome is heavily transcribed from numerous promoters, giving posttranscriptional events a critical role in determining the quantity and sizes of accumulating RNA species. The major events reviewed here are RNA editing, which restores protein conservation or creates correct open reading frames by converting C residues to U, RNA splicing, which occurs both in cis and trans, and RNA cleavage, which relies on a variety of exoribonucleases and endoribonucleases. Because the RNases have little sequence specificity, they are collectively able to remove extraneous RNAs whose ends are not protected by RN...
The chloroplast is a compartment unique to photosynthetic eukaryotes. It and other members of the... more The chloroplast is a compartment unique to photosynthetic eukaryotes. It and other members of the plastid family carry out a remarkable range of activities. Explorations of photosynthesis-related phenomena have stimulated biochemical, biophysical, molecular and genetic approaches to understanding the proplastid-to-chloroplast conversion and the regulation of chloroplast processes. Much of this regulation is exerted by nuclear gene products, which often participate in plastid gene expression. Concerted exploitation of genomic resources promises a deeper understanding of these regulatory factors and more effective genetic engineering based on chloroplast transformation. Here, we review recent progress, drawing attention to maize, whose unique attributes and new resources hold special promise for explorations of chloroplast biogenesis. Glossary NPPs: nucleus-encoded plastid proteins.
The regulation of chloroplast translation by nuclear gene products makes a major contribution to ... more The regulation of chloroplast translation by nuclear gene products makes a major contribution to the control of chloroplast gene expression, but the underlying mechanisms are poorly understood. We describe a pentatricopeptide repeat (PPR) protein in maize, ATP4, that is necessary for translation of the chloroplast atpB open reading frame. We demonstrate that ATP4 associates in vivo with sequences near the 5' end of the unusually long 5' UTR of the atpB/E mRNA, that it facilitates ribosome association with this mRNA, and that it is required for accumulation and activity of the chloroplast ATP synthase. ATP4 is multifunctional, in that it also enhances atpA translation and is required for accumulation of specific processed atpF and psaJ transcripts. ATP4 belongs to a sub-class of PPR proteins that include a small MutS-related (SMR) domain. SMR domains had previously been associated primarily with DNA-related functions, but our findings imply that at least some PPR-SMR proteins can act on RNA. ATP4 is orthologous to the Arabidopsis protein SVR7, but the phenotypes of atp4 and svr7 mutants suggest that the functions of these orthologs have not been strictly conserved.
The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeating motif that is found in... more The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeating motif that is found in animal, fungal, and plant proteins. The PPR protein family is particularly large in plants, where the majority of family members are predicted to be targeted to mitochondria or chloroplasts. PPR proteins are believed to fall into the larger family of helical repeat proteins, which typically bind macromolecules through a surface formed by the stacking of consecutive helical repeating units. Prior findings implicate several PPR proteins in organellar RNA metabolism, but the biological functions of few PPR proteins have been explored and in no case has a direct substrate been definitively identified. We present a characterization of the maize nuclear gene ppr2, which encodes a chloroplast PPR protein. PPR2 is found in large, heterogeneous protein complexes in the chloroplast stroma, some of which may be associated with RNA. Null ppr2 mutants have albino leaves and lack plastid rRNA and translation products. Plastid rRNAs are absent in both dark- and light-grown leaf tissues, indicating that their absence does not result from photo-oxidative damage. The population of plastid transcripts in ppr2 mutants is similar to that in other maize mutants lacking plastid ribosomes, and no ppr2-specific defects in plastid RNA metabolism have been detected. Taken together, the results suggest that ppr2 functions in the synthesis or assembly of one or more component of the plastid translation machinery.
The pentatricopeptide repeat (PPR) is a degenerate 35–amino acid repeat motif that is widely dist... more The pentatricopeptide repeat (PPR) is a degenerate 35–amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown ...
Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as ... more 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...
The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNA... more The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNAs and for the processing of the petD mRNA from a polycistronic precursor. In order to understand the biochemical role of the crp1 gene product and the interconnections between chloroplast translation and RNA metabolism, the crp1 gene and cDNA were cloned. The predicted crp1 gene product (CRP1) is related to nuclear genes in fungi that play an analogous role in mitochondrial gene expression, suggesting an underlying mechanistic similarity. Analysis of double mutants that lack both chloroplast ribosomes and crp1 function indicated that CRP1 activates a site-specific endoribonuclease independently of any role it plays in translation. Antibodies prepared to recombinant CRP1 were used to demonstrate that CRP1 is localized to the chloroplast stroma and that it is a component of a multisubunit complex. The CRP1 complex is not associated detectably with either chloroplast membranes or chloroplast ribosomes. Models for CRP1 function and its relationship to other activators of organellar translation are discussed.
data). Because the CAFs and CRS1 have no significant similarity outside of their repeated domains... more data). Because the CAFs and CRS1 have no significant similarity outside of their repeated domains, it seems likely that these domains contribute to the splicing functions of these proteins, perhaps by binding RNA. The prokaryotic homologs of these domains have been
Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are ... more Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are related to the tetratricopeptide repeat (TPR). Most characterized PPR proteins mediate specific post-transcriptional steps in gene expression in mitochondria or chloroplasts. However, little is known about the structure of PPR proteins or the biochemical mechanisms through which they act. Here we establish features of PPR protein structure and nucleic acid binding activity through in vitro experiments with PPR5, which binds and stabilizes a chloroplast tRNA precursor harboring a group II intron. Recombinant PPR5 was shown to be monomeric by analytical ultracentrifugation and gel filtration. Circular dichroism spectroscopy showed that PPR5 has a high content of α helices, as predicted from the similarity between PPR and TPR motifs. PPR5 and another PPR protein, CRP1, bind with high affinity to single-stranded RNA, but bind poorly to single-stranded DNA or to double-stranded RNA or DNA. A ...
Proceedings of the National Academy of Sciences, 1991
We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon.... more We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and ...
Proceedings of the National Academy of Sciences, 2013
Significance Transcripts in plant organelles are altered by conversion of cytidines to uridines i... more Significance Transcripts in plant organelles are altered by conversion of cytidines to uridines in a process termed RNA editing. Members of two protein families have been identified in the plant editosome, but its complete composition is unknown. Now a unique protein that contains an RNA recognition motif has been found to be essential for editing of multiple plastid transcripts in both Arabidopsis and maize. Phylogenetic analysis indicates that this protein belongs to a sub-family of RNA recognition-motif proteins predominantly predicted to be targeted to organelles and that are thus likely to play roles in organelle RNA metabolism.
The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large fami... more The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.
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Papers by Alice Barkan