Modification of Pentatricopeptide Repeat Proteins to Bind Defined Target RNA Sequences

Description:

Background

The manipulation of plant genomes is being used to modify traits of agronomic relevance and to exploit plants as biofactories for the production of desired products, such as biofuels, bioplastics or pharmaceuticals. Transplastomics, the introduction of transgenes into plastid genomes, has several key advantages over nuclear genomic transformation, including high expression levels, stable expression, and transgene containment (maternal-only inheritance of the plastid genome offers a natural barrier to the inadvertent spread of genetically modified plants).  However, high level expression of foreign proteins in plastids often compromises plant growth and fertility, necessitating the development of regulatory systems to prevent transgene expression until shortly before harvest. In addition, it can be advantageous to express foreign proteins in storage organs such as fruits and tubers, but low level gene expression in non-green plastids limits such applications. Recent advances from the laboratory of Prof. Barkan in understanding mechanisms by which PPR proteins stimulate the expression of specific chloroplast genes, together with the recent elucidation by Profs. Barkan and Small of an amino acid “code” by which PPR proteins bind specific RNAs, provide a path to overcome these challenges.

Technology summary

Pentatricopeptide Repeat (PPR) proteins are a large family of RNA-binding proteins particularly prevalent in plants. These proteins are encoded in the nucleus, but function in chloroplasts and mitochondria to regulate gene expression at the post-transcriptional level (RNA stability, translation, splicing, editing, etc.)1. Professor Barkan’s laboratory has defined the cis-elements bound by particular PPR proteins, and elucidated the mechanisms by which these proteins activate the expression of specific plastid genes2,3,4. Many endogenous chloroplast genes go virtually unexpressed in the absence of cognate PPR proteins. Therefore, regulated expression of PPR proteins can be used to control the expression of plastid transgenes with suitable flanking cis-elements.

Recently, UO Prof. Alice Barkan, in collaboration with Prof. Ian Small at University of Western Australia deciphered an amino acid “code” that determines which RNA sequences are bound by a particular PPR protein 5.  Their work showed that PPR proteins bind RNA via a modular 1 repeat-1 nucleotide recognition mechanism, in which the identities of specific amino acids in each repeat dictate which nucleotide will be bound. This information has allowed them to engineer novel PPR proteins that bind RNA sequences of their choice5, thus paving the way towards using these proteins as regulators of any gene (endogenous or artificial) encoded in mitochondria and chloroplasts. Given the high specificity of PPR proteins and the fact that most plastid genes are highly dependent on them for expression, PPR proteins provide a powerful tool for the regulation of plant organelle gene expression. In particular, the regulated transcription of engineered PPR genes in the nucleus in conjunction with cognate binding sites near plastid transgenes can be used to control transgene expression without disrupting the expression of endogenous genes. Additionally, Prof. Barkan is currently characterizing how the precise location of PPR protein binding on an RNA molecule can determine the level of expression of the associated gene, which will provide a way to fine-tune the expression level of any plastid gene or transgene of interest.

Patent status

PCT patent application filed April 2013 covering the use of the PPR-RNA binding code for gene regulation.

 

References

1- Barkan A, Small I. Pentatricopeptide Repeat Proteins in Plants. Annu Rev of Plant Biol. 2013, in press.

2- Pfalz J, Bayraktar OA, Prikryl J, and Barkan A. (2009) Site-specific binding of a PPR protein defines and stabilizes 5’ and 3’ mRNA termini in chloroplasts. EMBO J. 28: 2042-2052.

3- Prikryl J, Rojas M, Schuster G, Barkan A. Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein. Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):415-20.

4- Zhelyazkova  P, Rojas M, Hammani K, Voelker R, Vargas-Suarez M, Börner T and A Barkan. (2012) Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. Nucl. Acids Res. 40:3092-3105.

5- Barkan A, Rojas M, Fujii S, Yap A, Chong YS, Bond CS, Small I. A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet. 2012;8(8):e1002910.

 

Patent Information:
Category(s):
Research Tools
For Information, Contact:
Jim Deane
University of Oregon
jdeane@uoregon.edu
Inventors:
Alice Barkan
Ian Small
Sota Fujii
Charles Bond
Margarita Rojas
Yee Seng Chong
Keywords: