Build or destroy? Investigating how plants connect protein synthesis and degradation during mRNA translation to regulate protein quality

Secondary Supervisor(s): Dr Saverio Brogna

University of Registration: University of Birmingham

BBSRC Research Themes: Sustainable Agriculture and Food (Plant and Crop Science)

Project Outline

Proteins are essential components of cells, and the proteome - i.e., the complete cohort of proteins and their modification status - must be effectively regulated to maintain cellular function. A critical contributory step to proteome function is protein synthesis, where mRNAs are decoded and translated into polypeptides by the ribosome. However, problems can arise during this process, leading to the production of proteins that contain errors that negatively impact cellular function. Consequently, defective mRNAs and proteins must be recognised and destroyed, and dedicated machineries exist to facilitate this. Despite their central importance for ensuring proteome homeostasis (proteostasis), mechanisms controlling co-translational mRNA and protein quality in the plant kingdom are still largely unknown. This represents a major ‘black-box’ in the field of plant cell biology, since precise protein translation is essential for coordinating growth, development and resistance to environmental stresses that negatively impact plant productivity and survival. Increasing our understanding of the breadth of mechanisms employed by plants to ensure effective mRNA translation has strong potential to identify novel targets for manipulating traits of agronomic importance and could facilitate the development of improved crop varieties with enhanced performance.

We recently identified a family of E3 ubiquitin ligases in Arabidopsis that function at the interface of mRNA translation and protein destruction. Here, we will investigate the hypothesis that these ligases are components of an expanded and plant-specific “toolkit” that provides dynamic and stress-responsive functions in translational quality control.

Using a range of cutting edge molecular and biochemical techniques, we will decipher roles for these regulators in controlling global translation. There are several key areas where a PhD candidate would focus their investigations:

• Defining how these E3 ligases dynamically associate with ribosomes, proteasomes and other cellular machineries to form ‘translasomes’ that act as quality surveillance hubs.
• Characterising their direct proteolytic and mRNA targets.
• Determining how they collectively contribute to the control of global a mRNA translation and protein degradation, particularly within the context of environmental stress.
• Defining their roles in the degradation of stalled or misfolded proteins that form when translational errors arise.

The majority of our work is carried out in the genetic model plant Arabidopsis, with the eventual aim of this research being to translate key findings into crop species, such as barley or rice. The research will be largely molecular based and will also include ‘omics’ approaches. The PhD candidate will therefore gain expertise in a wide range of cutting edge and transferable techniques.

Iron-ing out a role for the Endoplasmic Reticulum in plant metal homeostasis

Secondary Supervisor(s): Dr Juliet Coates

University of Registration: University of Birmingham

BBSRC Research Themes: Sustainable Agriculture and Food (Plant and Crop Science)

Project Outline

Iron is an essential nutrient for almost all life on earth. As a result, organisms have evolved sophisticated ways of regulating iron sensing, uptake, and transport, to ensure an optimal supply of this metal is maintained. Plants typically extract iron from the soil and are able to sense when they are iron-starved, which leads to a rapid increase in the expression of iron uptake transporters via a transcriptional cascade. Although many of the molecular players are known, mechanisms controlling iron homeostasis require further study.

We recently discovered an Arabidopsis mutant that displays a constitutive iron starvation response - that is, many of the key iron signalling, uptake, and transport genes are up-regulated in this mutant even under “normal” conditions. The mutated gene encodes for an E3 ligase that is found in the membrane of the endoplasmic reticululum (ER), where it is proposed to be involved in the ubiquitination and degradation of proteins. We hypothesise that in the mutant, one (or several) of its degradation targets are stabilised, which then "tricks" the plant into thinking it is iron deficient even when it is not. As such, this E3 ligase may represent a previous unknown regulator of iron homeostasis, which has important implications for plant growth, development, and response to stress.

This PhD project seeks to investigate this hypothesis further using a range of physiology, molecular and biochemistry approaches. You will investigate internal iron levels and transport, and examine how wild type, mutant and overexpressing lines of this E3 ligase respond to changes in external iron availability, by growing on iron deficient and iron-replete media and monitoring growth and survival. You will use a combination of RNAseq, proteomic analyses, and Western blotting to examine gene expression and protein dynamics in response to external iron availability and other stresses, and you will also investigate how iron levels control the ubiquitination activity of the E3 ligase itself. The overall outcome of the project will be a better understanding of how the ER - via the action of a protein degradation mechanism - contributes to iron homeostasis in plants. This has strong potential for identifying new targets that could be manipulated for agronomic benefit.