Sandbox WWC7

From Proteopedia

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,^[1] translation,^[2] and organelle biogenesis.^[3] 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 . These hairpin structures accumulate to form an (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 Zea mays. PPR10 is often used as a model PPR protein.^[2]

Function

In the Zea mays plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and (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.^[2]

Mechanism

The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.^[4] Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.^[5]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.^[4] "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 EC-SY-SAX 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.^[6][7]

RNA Stabilization

PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.^[2]

Translation Enhancement

The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.^[2]

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.^[8] 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.^[5]

Limitations

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.^[5] 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.^[5]

References