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Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' ~~which~~ is often used as a model PPR protein.<ref name = "translation"/>

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, most known PPR proteins are found in plants, and they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of <span class="plainlinks">[https://en.wikipedia.org/wiki/Maize ''Zea mays'']</span>. PPR10 is often used as a model PPR protein.<ref name = "translation"/>

==Function==

In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these ~~mRNAs~~ are translated.<ref name = "translation"/>

In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==

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This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by <span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX]</span> has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

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==Synthetic Applications==

Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.

Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

===Limitations===

As PPR proteins themselves were only discovered recently, there is still a great deal to be learned ~~about them~~ before ~~this~~ becomes a viable technology. . .

As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

==References==

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-Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
+Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, most known PPR proteins are found in plants, and they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of <span class="plainlinks">[https://en.wikipedia.org/wiki/Maize ''Zea mays'']</span>. PPR10 is often used as a model PPR protein.<ref name = "translation"/>
 <Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />
 ==Function==
-In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these mRNAs are translated.<ref name = "translation"/>
+In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>
 ==Mechanism==
@@ Line 11: / Line 11: @@
 This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.
 While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by <span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX]</span> has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>
@@ Line 21: / Line 22: @@
 ==Synthetic Applications==
-Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.
+Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>
 ===Limitations===
-As PPR proteins themselves were only discovered recently, there is still a great deal to be learned about them before this becomes a viable technology. . .
+As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>
 ==References==
 {{Reflist}}