The Arabidopsis NOT4A E3 ligase coordinates PGR3 expression to regulate chloroplast protein translation

Mark Bailey, Aiste Ivanauskaite, Julia Grimmer, Oluwatunmise Akintewe, Adrienne C. Payne, Ross Etherington, Anne-Marie Labandera, Rory Osborne, Marjaana Rantala, Sacha Baginsky, Paula Mulo, Daniel J. Gibbs

Preprint posted on 3 April 2020

Article now published in Nature Communications at

Talking to the chloroplast: a nuclear-encoded E3 ubiquitin ligase controls chloroplast translation via PPR proteins

Selected by Martin Balcerowicz

Background: The nucleus controls gene expression in the chloroplast

Besides their nuclear genome, plant cells harbour two additional sets of genes, those encoded by the mitochondria and chloroplast genomes. Many essential organelle genes have “migrated” to the nucleus over the course of evolution – in many plants, only around 5% of chloroplast proteins are actually encoded by the plastid genome. Nevertheless, the chloroplast harbours its own transcriptional and translational machinery for gene expression, which needs to be tightly coordinated with nuclear gene expression and chloroplast translocation of nuclear-encoded proteins to allow proper assembly of photosynthetic protein complexes.

Pentatricopeptide repeat (PPR) proteins represent one means by which the nucleus controls chloroplast gene expression – these RNA-binding proteins are nuclear-encoded, but translocate into the chloroplast where they have manifold functions related to RNA maturation and metabolism – they affect RNA stability, editing and processing, but can also promote or reduce RNA translation (Barkan and Small, 2014). In the model plant Arabidopsis, approximately 450 PPR proteins are encoded in the genome, 41 of which have so far been shown to be targeted to the chloroplast (Rovira and Smith, 2019). Our understanding of how these PPR proteins are regulated is rather limited; in their preprint, Bailey et al. identify a novel regulator of a plastidic PPR protein and thereby contribute to our understanding of communication between nucleus and chloroplast.


Key findings: NOT4A regulates PGR3 RNA abundance and thereby chloroplast translation

NOT4, a peculiar protein harbouring both a RING ubiquitin ligase domain and an RNA recognition motif (RRM), is known to associate with ribosomes and functions in the control of mRNA and protein quality during the translation process in animals and yeast (Collart, 2016). Bailey et al. identified three homologues of NOT4 in Arabidopsis. By analysing loss-of-function mutants they found that one of them, NOT4A, is involved in chloroplast function. The not4a mutant is pale, develops more slowly and displays both reduced chlorophyll and reduced starch content compared to the wild type. RNA-seq and proteomics analyses underpinned this observation: many chloroplast-related proteins were less abundant in not4a, which caused disruption of several key protein complexes.

Intriguingly, while many nuclear-encoded proteins with low abundance in not4a also displayed low transcript amounts, this was not the case for plastid-encoded proteins. Especially proteins making up the small 30S subunit of the chloroplast ribosome were reduced in not4a, but the respective transcript levels were unchanged. This prompted the authors to investigate chloroplast translation. The not4a mutation had a similar effect as treatment with lincomycin, a selective inhibitor of plastidic translation. In addition, a puromycin labelling assay revealed strongly reduced levels of nascent proteins in not4a compared to wild-type chloroplasts. Taken together, these results suggest that compromised chloroplast translation is a likely cause for the observed photosynthetic deficiencies of not4a, as multiple key photosynthetic proteins are encoded in the chloroplast genome.

NOT4A does not harbour any chloroplast localisation signals and in agreement with this, a NOT4A-GUS fusion protein was not detected in plastidic protein fractions, suggesting that NOT4A affects translation from outside the chloroplast. Among the nuclear genes misregulated in not4a, PROTON GRADIENT REGULATED 3 (PGR3) was down-regulated to undetectable levels. It encodes a chloroplast-targeted PPR protein that is essential for translation of 30S ribosomal subunits (Rojas et al., 2018). Bailey et al. found that loss of PGR3 function mimicked the consequences of the not4A mutation, both in terms of the pale yellow, slow growth phenotype and in terms of displaying reduced chloroplast translation and 30S subunit depletion. Boosting PGR3 levels by expressing a PGR3-YFP fusion protein can rescue the not4a mutant phenotype, suggesting that PGR3 acts downstream of NOT4A in the control of plastidic translation. How NOT4A controls PGR3 levels is still unclear; Bailey et al. found that both the RING ubiquitin ligase domain as well as the RRM domain are essential for NOT4A function, but did not detect interaction between NOT4A and PGR3, nor did they observe effects on PGR3 protein turnover. It thus appears that NOT4A acts primarily on PGR3 transcript levels.

Figure 1: Visual phenotypes of not4a and pgr3 mutants. Images of 4-week-old not4a and pgr3 Arabidopsis mutants and their respective wild types. The bar indicates 1 cm (reproduced from Bailey et al., Figure 7e with permission of the authors).


Why I chose this preprint

Bailey et al. show us that a previously uncharacterised Arabidopsis E3 ubiquitin ligase contributes to the communication between two plant organelles – just in case we needed any further evidence that there are a lot of Arabidopsis genes whose functions remain enigmatic even today. In this context it is always exciting when a protein known from other model systems finally gets investigated in Arabidopsis and when we find out whether its role may or may not have diverged in plants.


Open questions/future directions

  1. Many nuclear-encoded genes are misregulated in the not4A mutant – do you have any indication whether this is mainly due to NOT4A’s role in nuclear gene regulation, or may this also involve retrograde signals from the chloroplast?
  2. It seems NOT4A does not regulate PGR3 stability, but rather affects its transcript levels. Is this most likely an indirect effect (e.g. by controlling stability of a transcription factor), or could it also involve binding/processing of the PGR3 mRNA by NOT4A?


References/further reading

Barkan, A. and Small, I. (2014). Pentatricopeptide Repeat Proteins in Plants. Annual Review of Plant Biology 65, 415–442.

Collart, M. A. (2016). The Ccr4‐Not complex is a key regulator of eukaryotic gene expression. Wiley Interdiscip Rev RNA 7, 438–454.

Rojas, M., Ruwe, H., Miranda, R. G., Zoschke, R., Hase, N., Schmitz-Linneweber, C. and Barkan, A. (2018). Unexpected functional versatility of the pentatricopeptide repeat proteins PGR3, PPR5 and PPR10. Nucleic Acids Res 46, 10448–10459.

Rovira, A. G. and Smith, A. G. (2019). PPR proteins – orchestrators of organelle RNA metabolism. Physiologia Plantarum 166, 451–459.

Tags: arabidopsis, chloroplast, rna, translation, ubiquitin ligase

Posted on: 4 May 2020


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Author's response

Mark Bailey and Daniel J. Gibbs shared

Your questions are on-point and the focus of ongoing work, but we’ll do our best to answer you…

  1. Many nuclear-encoded genes are misregulated in the not4A mutant – do you have any indication whether this is mainly due to NOT4A’s role in nuclear gene regulation, or may this also involve retrograde signals from the chloroplast?

1) Most likely we are seeing a combination of both. Our current studies aim to tease apart the effects due to loss of PPR versus loss of NOT4A expression, in addition to exploring the wider roles of all three NOT4 homologues in Arabidopsis. Watch this space…

  1. It seems NOT4A does not regulate PGR3 stability, but rather affects its transcript levels. Is this most likely an indirect effect (e.g. by controlling stability of a transcription factor), or could it also involve binding/processing of the PGR3 mRNA by NOT4A?

2) Both the RNA binding and ubiquitination activities of NOT4A are required to maintain PGR3 expression, so either of your latter two possibilities could be true.  We don’t think that this regulation is through proteolytic control of a transcription factor however, as we were able to transgenically express PGR3 in the not4a mutant under its native promoter.  Interestingly though, this ectopic PGR3 expression appeared to decouple it from NOT4A mediated influence, presumably due to missing regulatory elements (such as its 3´UTR), so we are examining this connection.

Studies in other organisms, have demonstrated the global influence of NOT4 on gene expression within, and independently of, the CCR4 NOT complex. We’re excited to push forward our understanding of the expanded NOT4 homologues in plants, exploring conserved and divergent activities, such as the chloroplast-specific function featured in our manuscript.


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