Progress 09/01/06 to 08/31/09
Outputs
OUTPUTS: This project focused on a large protein family containing a motif called the pentatricopeptide repeat (PPR) motif, which plays broad and essential roles in mitochondrial and chloroplast gene expression. Prior studies suggested that most PPR proteins mediate specific post-transcriptional steps in organellar gene expression, and that they do so via direct interaction with RNA. Despite their key roles as integrators of nuclear and organellar functions, little is known about the biochemical mechanisms through which PPR proteins act. The goal of this project was to establish a biochemical framework for understanding PPR proteins and their mode of RNA recognition, through in vitro experiments with several chloroplast-localized PPR proteins in maize. We made considerable progress toward this goal. (i) We developed methods to express and purify several recombinant PPR proteins in soluble form (CRP1, PPR5, and PPR10). This was an achievement because PPR proteins are notoriously prone to aggregation. (ii) We used these proteins in in vitro assays to pinpoint the precise RNA sequences to which they bind, and to demonstrate several fundamental biochemical properties of PPR proteins. The assays included CD-spectroscopy to monitor protein folding and helical content, gel mobility shift assays to monitor RNA binding activity, and assays to monitor protein-induced RNA unfolding. (iii) Funding from another source allowed the simultaneous genetic analysis of these same proteins; in this way, we linked these biochemical activities with the direct in vivo functions of each of these proteins. Together, this body of data provided what we feel to be the most incisive mechanistic data available for members of the PPR family. Our findings have broad implications for PPR mechanisms, and for mechanisms of organellar gene expression, as outlined in Outcomes/Impacts. PARTICIPANTS: Alice Barkan was the PI, and was actively involved in the design and interpretation of experiments. Rosalind Williams-Carrier, a research assistant in the lab, took the leading role in the PPR5 biochemical work. Postdoctoral fellow Jeannette Pfalz did most of the work with PPR10. Jana Prikryl, a Ph.D. student, explored the RNA helix destabilizing activity of PPR5. Jana also contributed to defining the precise binding site of PPR10 and providing data that supports the view that many other PPR proteins have a similar mode of action. Jana presented a poster on this project at the Gordon Research Conference on Mitochondria and Chloroplasts. Omer Ali Bayraktar- Ph.D. student on rotation in the lab. Demonstrated the RNA binding specificity of recombinant PPR10. Christian Schmitz-Linneweber, a former postdoctoral fellow in the lab, initiated the genetic analysis of PPR5. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
(i) We showed that CRP1, PPR5, and PPR10 each bind RNA with high-affinity, providing direct evidence that PPR proteins generally function as RNA binding proteins. (ii) We showed that these proteins bind all types of nucleic acids (DNA, RNA, double-stranded or single-stranded), but they have a very strong preference for single-stranded RNA. (Williams-Carrier et al, RNA). We believe that this preference underlies their ability to disrupt RNA hairpins (see below). (iii) We used CD-spectroscopy to show that PPR5 is composed largely of alpha helices. Although PPR tracts are predicted to adopt a helical-repeat superhelical structure, our data remains the only experimental structural data for the PPR family. (Williams-Carrier et al, RNA) (iv) We determined the precise RNA binding site and in vivo function of PPR5. Site-specific binding of PPR5 to an endonuclease-sensitive site in the trnG intron protects that site from cleavage, thereby promoting accumulation of the unspliced (and thus the spliced) trnG molecule. (Beick et al, MCB; Williams-Carrier et al, RNA) (v) We determined the precise RNA binding sites and in vivo function of PPR10. PPR10 binds to two intergenic RNA regions on polycistronic RNAs, and stabilizes the adjacent protein-coding regions by blocking 5' and 3' exonucleases. (Pfalz et al, EMBO) (vi) We provided evidence that both PPR5 and PPR10 function as site-specific RNA unfoldases, and that this activity has important consequences for the way they influence gene expression. (Manuscript in preparation). (vii) We proposed a new model for the determinants of the processing and stability of chloroplast RNAs that are based on these findings. We believe that the fundamental biochemical properties we described (the ability to bind a long tract of single-stranded RNA with great sequence specificity, the ability to block RNA degradation by exonucleases, and the ability to function as site-specific RNA unfoldases) underlie many of the apparently diverse biological functions of PPR proteins in both mitochondria and chloroplasts. I am planning to write a review article that describes these ideas. These findings were presented at several conferences: the Gordon Research Conference on Mitochondria and Chloroplasts, the Maize Genetics Conference, the Plant and Animal Genome conference, and the International Conference on Plant Mitochondria.
Publications
- Williams-Carrier RE, Kroeger T, and Barkan A. (2008) Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand. RNA, 14: 1930-41.
- Beick S, Schmitz-Linneweber, Williams-Carrier R, Jensen B, and Barkan A. (2008) The pentatricopeptide repeat protein PPR5 stabilizes a specific tRNA precursor in maize chloroplasts. Molec. Cell. Biol., 28: 5337-47.
- Pfalz J, Bayraktar OA, Prikryl J, and Barkan A. (2009) Site-specific binding of a PPR protein defines and stabilizes 5 prime and 3 prime mRNA termini in chloroplasts. EMBO J. 28: 2042-2052.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: This project explores the basis for RNA recognition by a large protein family containing a motif called the pentatricopeptide repeat (PPR) motif. Prior studies suggest that most PPR proteins mediate specific post-transcriptional steps in organellar gene expression, and that they do so via direct interaction with RNA. Despite their key role as integrators of nuclear and organellar functions, little is known about the biochemical mechanisms through which PPR proteins act. The goal of this project is to establish a biochemical framework for understanding PPR proteins and their mode of RNA recognition, through in vitro experiments with several maize PPR proteins. During Year 1, we established methods to express two PPR proteins, CRP1 and PPR5, as soluble recombinant proteins. We demonstrated that both proteins bind all nucleic acids, but have strong preference for single-stranded RNA. We also pinpointed the PPR5 binding site to a specific 50 nt sequence. Our progress in Year 2 is summarized below. i) We found that the absence of PPR5 in vivo results in the cleavage of the trnG precursor at a specific site. Mapping of this cleavage site revealed that it corresponds precisely to the sequence bound by recombinant PPR5 in vitro. Together, the genetic and biochemical data provide the first mechanism for how a PPR protein influences gene expression: site-specific binding of PPR5 to an endonuclease-sensitive site in the trnG intron protects that site from cleavage, thereby promoting accumulation of the unspliced (and thus the spliced) trnG molecule. ii) We found that the PPR5 binding site includes a stable RNA hairpin that sequesters the "EBS1" element, which must pair with a complementary sequence in exon 1 to define the 5' splice junction. This led us to speculate that PPR5 promotes trnG expression in two ways: by stabilizing the unspliced precursor and by destabilizing the hairpin within its binding site. Preliminary data support the idea that PPR5 destabilizes the RNA hairpin in vitro. This is a significant finding, because the ability of a PPR protein to unwind an RNA duplex has implications for how other PPR proteins influence gene expression. ii) We determined the function and RNA sequence recognized by a different PPR protein, PPR10. PPR10 is necessary to stabilize transcripts from each of two chloroplast loci: atpH and psaJ. PPR10 binds in vivo to the 5' UTR of certain atpH transcripts and to the 3' UTR of certain psaJ transcripts. The RNAs missing in ppr10 mutants correspond exactly to those whose termini map to these PPR10 interaction sites. These results build on the work with PPR5, demonstrating that binding of a PPR protein to either a 5' UTR or a 3' UTR can protect an RNA from the rate-limiting cleavages that initiate RNA decay. The two RNA elements bound in vivo by PPR10 share a consensus sequence that we hypothesized is the PPR binding site. We were able to generate soluble recombinant PPR10, and we found that it binds with specificity in vitro to RNAs harboring this consensus sequence. PARTICIPANTS: Alice Barkan is the PI, and is actively involved in the design and interpretation of experiments. Rosalind Williams-Carrier, a research assistant in the lab, has taken the leading role in the PPR5 biochemical work. Postdoctoral fellow Jeannette Pfalz did the work with PPR10. Jana Prikryl, a Ph.D. student, recently joined the project and is exploring the RNA helix destabilizing activity of PPR5. Jana attended the Gordon Research Conference on Mitochondria and Chloroplasts this past summer, and presented a poster on this project. Christian Schmitz-Linneweber, a former postdoctoral fellow in the lab, initiated the genetic analysis of PPR5. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We now have more information concerning the specific RNA sequences recognized by PPR5 and PPR10 than is available for any other PPR protein. PPR proteins clearly have exquisite specificity for particular RNA sequences, but nothing is known about the biochemical or structural basis of this specificity. We are now poised to be able to use recombinant PPR5 and PPR10 to explore these issues. Ultimately, we hope to be able to design PPR proteins to target any desired RNA sequence in the cell.
Publications
- Williams-Carrier RE, Kroeger T, and Barkan A. (2008) Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand. RNA, 14: 1930-41.
- Beick S, Schmitz-Linneweber, Williams-Carrier R, Jensen B, and Barkan A. (2008) The pentatricopeptide repeat protein PPR5 stabilizes a specific tRNA precursor in maize chloroplasts. Molec. Cell. Biol., 28: 5337-47.
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Progress 09/01/06 to 08/31/07
Outputs
This project explores the basis for RNA recognition by a large protein family containing an RNA binding motif called the pentatricopeptide repeat (PPR) motif. PPR proteins are found in all eucaryotes but the family is dramatically expanded in plants. Recent observations implicate the PPR family as playing a particularly central and broad role in modulating the expression of mitochondrial and chloroplast genes. Prior studies suggest that most PPR proteins mediate specific post-transcriptional steps in organellar gene expression, and that they do so via direct interaction with RNA. Despite their key role as integrators of nuclear and organellar functions, very little is known about the biochemical mechanisms through which PPR proteins act. The goal of this project is to establish a biochemical framework for understanding PPR proteins and their mode of RNA recognition, through in vitro experiments with three maize PPR proteins. The in vivo functions and native RNA ligands
of each of these proteins were established previously, ensuring that the interactions studied in vitro are physiologically-relevant. Recombinant proteins will be analyzed to establish biophysical features such as helical content and overall symmetry/asymmetry, and to decipher the mechanisms by which the bind RNA. Year 1 Progress PPR proteins are notoriously difficult to generate as untagged, purified recombinant proteins. We optimized conditions for the expression and purification of two recombinant PPR proteins, CRP1 and PPR5. We used those proteins in the following biophysical and biochemical assays. a) Circular dichroism spectroscopy and analytical ultracentrifugation with purified recombinant PPR5 showed it to have a high helical content and to be an elongated monomer. These are the first biophysical assays of a PPR protein and they support structural predictions made by extrapolation from the related TPR motif. b) We showed that recombinant CRP1 and PPR5 both bind nucleic acids
in vitro. We showed further that they both have a strong preference for single-stranded RNA, relative to single-stranded DNA or double-stranded RNA/DNA. These are the first such data to be reported for PPR proteins. c) We showed that recombinant PPR5 binds with strong specificity to a 50-nt segment of the intron in the chloroplast trnG-UCC gene (PPR5's native ligand). Nothing is known about how PPR proteins recognize specific RNA sequences. We are now poised to be able to use recombinant PPR5 and this trnG intron sequence to explore the biochemical and structural basis of this sequence-specificity. These findings have been described in a manuscript that is currently under revision for the journal RNA.
Impacts
Elucidation of the mechanism of RNA recognition by plant-specific RNA binding proteins such as PPR proteins will increase our understanding of the control of gene expression in plants, and may result in the ability to engineer proteins that have novel RNA specificities and that can be used to control the expression of endogenous or introduced genes.
Publications
- No publications reported this period
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