Arabidopsis NOT4 E3 ligases: key regulators of TOR signalling and co-translational protein quality control

Schwarze, Maximillian Andreas ORCID: 0000-0002-0255-5427 (2025). Arabidopsis NOT4 E3 ligases: key regulators of TOR signalling and co-translational protein quality control. University of Birmingham. Ph.D.

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Abstract

Protein synthesis and degradation are both essential for regulating proteome homeostasis and cellular function. In eukaryotes the predominant mechanism for regulated protein degradation is via the ubiquitin proteasome system (UPS), where substrates are marked for degradation through the addition of a polyubiquitin chain by enzymes called E3 ubiquitin ligases. The UPS is important for degrading proteins when they are no longer needed, but also plays a key role in clearing misfolded proteins that arise during stress or in response to translational errors as proteins are synthesised. In yeast and metazoans, the NOT4 E3 ligase has been shown to play a role in co-translational mRNA and protein quality control to limit translational errors, but the presence and functions of NOT4-like proteins in plants has not been investigated.

Three putative NOT4 E3 ligases (AtNOT4A, B and C) were identified in Arabidopsis and shown to contain the unique and characteristic combination of a RING domain and RNA recognition motif (RRM), which places their function at the interface of proteolysis and RNA biology. Phenotypic and RNA-seq analyses of single and combination Atnot4 mutants revealed redundant functions for these proteins in controlling plant growth – likely due to energy deficiencies – and regulating the expression of genes linked protein translation. Further analyses showed that plants lacking functional AtNOT4s have increased global translation rates, accumulate polyubiquitinated proteins, and are sensitive to protein-folding elicitors, which is indicative of increased error rates during protein biogenesis.

AtNOT4s had previously been identified as targets of the AtTOR kinase, a master regulator in energy- and nutrient-responsive translation. Given the growth and translational phenotypes observed in Atnot4 mutants, an assessment of their relationship to AtTOR signalling was undertaken. Atnot4 mutants were found to have increased basal rates of AtTOR activity, which may explain the increased rates of translation in these mutants. Moreover, biochemical, plant growth, and RNA-seq analyses showed that Atnot4 mutants are sensitive to chemical inhibitors of the AtTOR signalling cascade, suggesting that they are required for normal AtTOR functions.

In summary, the data presented in this thesis describe for the first time a role for plant NOT4-like proteins in regulating protein translation linked to AtTOR signalling, suggesting they function as part of a conserved co-translational surveillance mechanism that coordinates and safeguards protein production. This has important implications for our understanding of proteostatic quality control mechanisms across kingdoms, whilst also identifying new potential targets for enhancing plant performance through improving translational fidelity under normal and stress conditions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):