Targeted cleavage of nad6 mRNA induced by a modified pentatricopeptide repeat protein in plant mitochondria

Catherine Colas Des Francs-Small,Lilian Vincis Pereira Sanglard,Ian Small

doi:10.1038/s42003-018-0166-8

Catherine Colas Des Francs-Small, Lilian Vincis Pereira Sanglard + Show 1 more

Open Access

https://doi.org/10.1038/s42003-018-0166-8

Copy DOI

Abstract

Mitochondrial genes encode key components of the cellular energy machinery, but their genetic analysis is difficult or impossible in most organisms (including plants) because of the lack of viable transformation approaches. We report here a method to block the expression of the mitochondrial nad6 gene encoding a subunit of respiratory complex I in Arabidopsis thaliana, via the modification of the specificity of the RNA-binding protein RNA PROCESSING FACTOR 2 (RPF2). We show that the modified RPF2 binds and specifically induces cleavage of nad6 RNA, almost eliminating expression of the Nad6 protein and consequently complex I accumulation and activity. To our knowledge, this is the first example of a targeted block in expression of a specific mitochondrial transcript by a custom-designed RNA-binding protein. This opens the path to reverse genetics studies on mitochondrial gene functions and leads to potential applications in agriculture.

Highlights

Mitochondrial genes encode key components of the cellular energy machinery, but their genetic analysis is difficult or impossible in most organisms because of the lack of viable transformation approaches
We searched for other similar sequences in the A. thaliana mitochondrial transcriptome, and found that the sequence at position + 349–365 in the coding sequence of nad[6] has only 3 differences to the predicted RNA PROCESSING FACTOR 2 (RPF2)-binding site in the cox[3] transcript
The 5′–3′ exonuclease activity is unknown from plant mitochondria[31]; we suggest that the initial cleavage is 55 nt 3′ of the RFP-nad6-binding site, followed by 3′–5′ exonucleolytic degradation, which is inhibited from extending further by the bound pentatricopeptide repeat (PPR) protein

Summary

Introduction

Mitochondrial genes encode key components of the cellular energy machinery, but their genetic analysis is difficult or impossible in most organisms (including plants) because of the lack of viable transformation approaches. We report here a method to block the expression of the mitochondrial nad[6] gene encoding a subunit of respiratory complex I in Arabidopsis thaliana, via the modification of the specificity of the RNA-binding protein RNA PROCESSING FACTOR 2 (RPF2). We show that the modified RPF2 binds and induces cleavage of nad[6] RNA, almost eliminating expression of the Nad[6] protein and complex I accumulation and activity To our knowledge, this is the first example of a targeted block in expression of a specific mitochondrial transcript by a custom-designed RNA-binding protein. Restorer-of-fertility proteins form a clade within the P-class PPR proteins that co-evolves with mitochondrial ORFs causing CMS4 They appear to block expression of specific mitochondrial ORFs by inducing RNA cleavage or degradation, or by preventing translation. We designed the RFL protein RNA PROCESSING FACTOR 2 (RPF2)[13] to bind a new RNA target, located within the coding sequence of nad[6], and show that the retargeting is successful

Methods

Results

Conclusion