School of Molecular Sciences

Postgraduate research profiles

Clement Boussardon

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Thesis

Molecular and functional characterization of the DYW1 protein in the chloroplast editing complex

Summary

Scientific summary:

RNA editing is a post-transcriptionnal process that converts, inserts or deletes nucleotides in many organisms. In plants, this modification consists in a deamination of a cytidine leading to a uridine. Thirty-four editing sites are found in chloroplasts and more than 500 in mitochondria of the model plant Arabidopsis thaliana.

Recently, the crr4 mutant of Arabidopsis has been found to have an editing defect in the ndhD-1 site of the ndhD transcript. In this site, an ACG is converted to an AUG to produce a translation initiation codon. The CRR4 protein is a specificity factor that binds to a sequence in the RNA but doesn’t seem to contain any catalytic activity that allows a deamination at this site. From this point, other proteins have been found to be involved in RNA editing.

All these proteins belong to the PPR (PentatricoPeptide repeat) proteins family. Within this family, different subgroup can be formed depending on their C-terminal end, named domain E (for extended) and domain DYW (corresponding to the conserved final amino acid triplet). The DYW domain focused our attention because it contains structures that are found in known cytidine deaminases in human and bacteria. It has been proposed that this domain has the catalytic activity for editing. Nevertheless, the CRR4 protein is part of PPR proteins that doesn’t contain any DYW domain. How is this protein able to edit without any DYW domain?

The DYW1 gene encodes a unique protein in Arabidopsis that contain a DYW domain without any PPR motifs. The hypothesis was that the DYW1 protein could somehow interact with PPR with only E domains to constitute an editing complex.

DYW1 gene has been mutated by EMS treatment. Among these mutants, the dyw1-1 mutant shows a change in the open reading frame leading to the apparition of a stop codon in the middle of the gene. We looked at editing site in the chloroplast (DYW1 is targeted in the chloroplasts) to see whether or not editing sites were affected. No editing sites were affected except the ndhD-1 site showing an abolition of RNA editing. To confirm that the editing defect was due to the mutation in DYW1, we complemented the dyw1-1 mutant with a modified strain of Agrobacterium tumefaciens containing the full DYW1 gene. By this method, editing of the ndhD-1 site is restored.

We already know that the CRR4 specificity factor is necessary for editing of this site. We have tested interaction between CRR4 and DYW1 in vivo by split-YFP. This test allowed us to show that these two proteins interact together in the chloroplast.

Sequence comparison showed that the PPR-E CRR4 is slightly truncated in its E domain and that DYW1 contains a really short E domain in N-terminal of the DYW domain. We hypothesized that DYW1 could be the missing part of CRR4 to form a complete PPR-DYW. To test it, a double mutant crr4-dyw1 was created and complemented by a fused form of CRR4 and DYW1, the junction is done between E domains of CRR4 and DYW. This fusion complemented perfectly the double mutant.

Despite these results, it is still impossible to prove that the DYW domain contains the enzyme allowing RNA editing. We performed many in vitro editing assays that have been unsuccessful. We assumed that the editing complex of DYW1 was incomplete.

Very recently, MORF proteins have been found to be also implicated in RNA editing. NdhD-1 editing site shows an abolition of editing in morf2 and morf9 mutants. These factors contain neither PPR motifs nor catalytic activity but are capable of binding cobalt ions. The new hypothesis proposed is that MORF2 and MORF9 recruit metal ions and give them to the DYW domain so that editing can occur.

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Non-scientific summary:

DNA is the main component of the genetic information in all living organisms. It’s a sequence of nucleobases A (Adenine), T (thymine), G (guanine) and C (cytosine). Thanks to the transcription process, DNA is copied in RNA, a molecule close to DNA excepting the fact that T are replaced by U (uracil). RNA is the necessary intermediate between DNA and protein production.

Editing is a natural phenomenon that rose in lots of organisms (Human and Plants included) that allows the modification of specific nucleobases C to U at defined sites in the RNA. This leads to a modification of the protein compared to the information contained in the DNA.

Editing is not well understood in plants. Nevertheless, by RNA binding, proteins called PPR are implicated in this process. The enzyme that catalyzes the reaction of editing is still unknown. The aim of my thesis is to investigate this molecular process. I have to identify the exact role of the DYW1 protein that might be the enzyme responsible for RNA editing reaction (hypothesis made because of its structure). DYW1 is unable to bind RNA so we assumed that another molecule (a PPR?) might be implicated as well.

My work shows that DYW1 is implicated in RNA editing at a specific site. Editing of this RNA is known to be dependant of a PPR protein, named CRR4. We proposed and demonstrated that DYW1 and CRR4 interact together to form what we call and editing complex. Nevertheless, it hasn’t been possible to show that DYW1 was the enzyme for editing. We assumed that the editing complex of DYW1 was incomplete. The recent discovery of new proteins (MORF) might allow us to demonstrate that DYW1 is the editing enzyme of the ndhD-1 site.

Why my research is important

PPR family is involved in post-transcriptionnal processes that are essential for almost all species. Within these processes, we find RNA editing that leads to the modification of specific C to U changes that leads to the alteration fo the protein sequence. It is not clear why natural selection favoured the apparition of RNA editing in plants. Most RNA editing modifications have a repairing function that restores amino-acids. But why would plants exclusively correct T to C “mutations”? It has been proposed that C to U editing is retained to neutralize T to C mutations that are unlikely to reverse. It is a hypothesis that has to be proven…

Moreover, in RNA editing partial editing was found for some sites with possibility of some unedited molecule to be translated. It is highly probable that two or more versions of some proteins are synthesized. That generates diversity in the proteome: with one unique gene, we can obtain many proteins.

Understanding of this process is necessary for understanding plants and genome behavior. RNA editing is energy consuming. Why the plant doesn’t repair directly its DNA sequence instead of each time modifying its RNA?

In a more practical way, understanding PPR and editing might be use for scientific purpose like targeted RNA modifications (RNA viruses, suppress RNA translation …). Of course the Holy Grail would be to cure genetic disease found in humans. Instead of trying to modify the information at the DNA level, why not trying to modify it at the RNA level? We are still far from this kind of awesome application… that’s why I’m working on understanding this process.

Funding

  • UPA and UWA Safety net top Up (09/2009 to 09/2012)
  • UPA and UWA Safety net top Up - Extension of scholarship (09/2012 to 03/2013)